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CN112940076B - FOXM1-derived peptides and vaccines containing the same - Google Patents

FOXM1-derived peptides and vaccines containing the same Download PDF

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Publication number
CN112940076B
CN112940076B CN202110182952.XA CN202110182952A CN112940076B CN 112940076 B CN112940076 B CN 112940076B CN 202110182952 A CN202110182952 A CN 202110182952A CN 112940076 B CN112940076 B CN 112940076B
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peptide
cancer
hla
peptides
foxm1
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CN112940076A (en
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山下祥子
引地哲郎
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Oncotherapy Science Inc
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Oncotherapy Science Inc
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Abstract

The present invention provides FOXM 1-derived epitope peptides having the ability to induce cytotoxic T cells. The invention also provides polynucleotides encoding the peptides, antigen presenting cells presenting the peptides, and cytotoxic T cells targeting the peptides, and methods of inducing antigen presenting cells or CTLs. The invention also provides compositions and pharmaceutical compositions containing them as active ingredients. In addition, the present invention provides methods of treating and/or preventing cancer, and/or preventing postoperative recurrence thereof, using the peptides, polynucleotides, antigen presenting cells, cytotoxic T cells, or pharmaceutical compositions of the present invention. Methods of inducing an immune response against cancer are also provided.

Description

FOXM 1-derived peptides and vaccines containing the same
The application is a divisional application of Chinese patent application of the application name of FOXM1 derived peptides and vaccines containing the same, which is 10/6/2016/201680057758.6/2016/date of application.
Technical Field
The invention relates to the field of bioscience, more particularly to the field of cancer treatment. In particular, the present invention relates to novel peptides effective as cancer vaccines, methods of using the peptides to treat and/or prevent tumors, and pharmaceutical compositions comprising the peptides.
Background
CD 8-positive cytotoxic T Cells (CTLs) are known to recognize epitope peptides derived from Tumor Associated Antigens (TAA) presented on Major Histocompatibility Complex (MHC) class I molecules expressed on the cell surface and then kill tumor cells. Since the discovery of the Melanoma Antigen (MAGE) family, many TAA(NPL1:Boon T,Int J Cancer 1993,54(2):177-80;NPL2:Boon T&van der Bruggen P,J Exp Med 1996,183(3):725-9). of these TAAs have been discovered by immunological methods are currently being developed clinically as immunotherapeutic targets.
Among several of these TAAs, epitope peptides that can be recognized by CTLs are identified, and their use in immunotherapy for various types of cancers (NPL3:Harris CC,J Natl Cancer Inst 1996,88(20):1442-55;NPL4:Butterfield LH et al.,Cancer Res 1999,59(13):3134-42;NPL5:Vissers JL et al.,Cancer Res 1999,59(21):5554-9;NPL6:van der Burg SH et al.,J Immunol 1996,156(9):3308-14;NPL7:Tanaka F et al.,Cancer Res 1997,57(20):4465-8;NPL8:Fujie T et al.,Int J Cancer1999,80(2):169-72;NPL9:Kikuchi M et al.,Int J Cancer 1999,81(3):459-66;NPL10:Oiso M et al.,Int J Cancer 1999,81(3):387-94). has been expected, to date, several clinical trials using these TAA-derived epitope peptides have been reported. Unfortunately, however, the response rate in many clinical trials is not high (NPL11:Belli F et al.,J Clin Oncol 2002,20(20):4169-80;NPL12:Coulie PG et al.,Immunol Rev 2002,188:33-42;NPL13:Rosenberg SA et al.,Nat Med 2004,10(9):909-15). and therefore, there remains a need to identify novel CTL epitope peptides that can be used in cancer immunotherapy.
As a result of the whole genome expression profile from the cDNA microarray targeting the 27,648 gene, FOXM1 (GenBank accession No.: nm_202003;Forkhead box M1) was identified and reported as gene (NPL14:Obama K et al.,Hepatology 2005,41(6):1339-48;NPL15:Yokomine K et al.,Int J Cancer 2010,126(9):2153-63;PTL1:WO2005/090603). whose expression was up-regulated in intrahepatic cholangiocarcinoma, non-small cell lung cancer and esophageal cancer tissues, on the other hand, FOXM1 expression levels in normal tissues were very low compared to these cancer tissues. In addition, when FOXM1 expression is inhibited, proliferation of medulloblastoma cells is inhibited (NPL 16: PRILLER M ET al, CLIN CANCER RES 2011,17 (21): 6791-801), FOXM1 is considered a gene involved in regulating proliferation of cancer cells. More specifically, FOXM1 is a TAA that many cancers repeatedly proliferate, and thus epitope peptides derived from FOXM1 are considered suitable for immunotherapy targeting cancer patients.
Recently, FOXM 1-derived HLA-A 02-restricted epitope peptides (PTL 2: WO 2009/025196) and HLA-A 24-restricted epitope peptides (PTL 3: WO 2010/095428) have been identified. The therapeutic effect of these peptides in cancer patients with HLA-A02 or HLA-A24 type could be expected, but not for other cancer patients.
[ Reference List ]
[ Patent literature ]
[PTL 1]WO2005/090603
[PTL 2]WO2009/025196
[PTL 3]WO2010/095428
[Non Patent Literature]
[NPL 1]Boon T,Int J Cancer 1993,54(2):177-80
[NPL 2]Boon T&van der Bruggen P,J Exp Med 1996,183(3):725-9
[NPL 3]Harris CC,J Natl Cancer Inst 1996,88(20):1442-55
[NPL 4]Butterfield LH et al.,Cancer Res 1999,59(13):3134-42
[NPL 5]Vissers JL et al.,Cancer Res 1999,59(21):5554-9
[NPL 6]van der Burg SH et al.,J Immunol 1996,156(9):3308-14
[NPL 7]Tanaka F et al.,Cancer Res 1997,57(20):4465-8
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Disclosure of Invention
The present invention relates to peptides that can induce CTLs that specifically react with FOXM 1-expressing cells. When these peptides form complexes with Human Leukocyte Antigens (HLA) and are presented to CD 8-positive T cells by Antigen Presenting Cells (APCs) that present the complexes on their surfaces, CTLs exhibiting peptide-specific cytotoxic activity are induced. FOXM 1-derived peptides having CTL inducibility (CTL inducibility) which have been identified so far are HLA-A 02-restricted peptides and HLA-A 24-restricted peptides, and cannot induce CTLs when antigen-presenting cells do not express these HLAs. Therefore, conventional peptides are not suitable for immunotherapy in subjects without these HLA. HLA-A33 is an allele common to Asians and HLA-A01 is an allele common to Cao K, et al, hum Immunol 2001,62 (9): 1009-30). It is desirable to administer an HLA-A33 restriction peptide to an HLA-A33 positive subject and an HLA-A01 restriction peptide to an HLA-A01 positive subject. Accordingly, the present invention relates to FOXM 1-derived peptides having CTL inducibility restricted to HLA-A33 or HLA-A 01. Based on the results disclosed herein, it has been demonstrated that the peptides of the present invention are epitope peptides capable of inducing a strong and specific immune response against FOXM1 and HLA-A33 or HLA-A01 expressing cells.
It is therefore an object of the present invention to provide FOXM 1-derived peptides capable of inducing CTLs in a HLA-A 33-or HLA-A 01-restricted manner. These peptides may be used to induce CTLs in vitro, ex vivo, or in vivo, or may be used for the purpose of administration to a subject for inducing an immune response against FOXM 1-expressing cancer cells. Preferred peptides are peptides ：SEQ ID NOs:1,2,3,6,7,11,12,17,18,20,22,24,26,32,33,36,39,41,42,45,46,48,49,50,52,53,55,56,57,58,59,60, and 61 comprising an amino acid sequence selected from the group consisting of nine or ten peptides, and even more preferred peptides are peptides SEQ ID NOs:1,2,3,6,7,11,12,17,18,20,22,24,26,32,33,36,39,41,42,45,46,48,49,50,52,53,55,56,57,58,59,60, and 61 selected from the group consisting of the amino acid sequences.
The peptides of the invention include peptides in which one, two or more amino acids are substituted, deleted, inserted and/or added, provided that the resulting modified peptide retains the CTL inducibility of the original peptide.
The invention also provides an isolated polynucleotide encoding any of the peptides of the invention. Like the peptides of the invention, these polynucleotides can be used to induce APCs with CTL inducibility and can be administered to a subject for inducing an immune response against FOXM 1-expressing cancer cells.
The invention also provides compositions comprising one or more types of peptides of the invention, one or more types of polynucleotides encoding one or more types of peptides of the invention, APCs of the invention, exosomes presenting the peptides of the invention, and/or CTLs of the invention. The composition of the present invention is preferably a pharmaceutical composition. The pharmaceutical composition of the present invention can be used for treating and/or preventing cancer, and preventing postoperative recurrence thereof. They may also be used to induce an immune response against cancer. When administered to a subject, the peptides of the invention are presented on the surface of APCs, and as a result, CTLs targeting the peptides are induced. It is therefore another object of the present invention to provide a composition for inducing CTLs, wherein the composition comprises one or more types of the peptides of the present invention, one or more types of polynucleotides encoding the one or more types of the peptides of the present invention, APCs of the present invention, and/or exosomes presenting the peptides of the present invention.
It is another object of the present invention to provide a method for inducing an APC having CTL inducibility, wherein the method comprises a step of contacting one or more types of peptides of the present invention with the APC, or a step of introducing a polynucleotide encoding any one of the peptides of the present invention into the APC.
The present invention also provides a method of inducing CTLs, comprising the step of co-culturing CD 8-positive T cells with APCs presenting a complex of an HLA antigen and a peptide of the present invention on the surface thereof, the step of co-culturing CD 8-positive T cells with exosomes presenting a complex of an HLA antigen and a peptide of the present invention on the surface thereof, or the step of introducing into CD 8-positive T cells a vector comprising a polynucleotide encoding each subunit of a T Cell Receptor (TCR) capable of binding to a peptide of the present invention presented by an HLA antigen on the cell surface. A preferred HLA antigen in the present invention is HLA-A33 or HLA-A01.
It is another object of the present invention to provide isolated APCs that present complexes of HLA antigens and the peptides of the present invention on their surface. The invention also provides isolated CTLs targeting the peptides of the invention. These APCs and CTLs can be used for immunotherapy of FOXM 1-expressing cancers. In the present invention, the cancer to be subjected to immunotherapy is, for example, a cancer present in a patient having a homozygote or a heterozygote of HLA-A33 or HLA-A 01. Thus, APC or CTL is also a cell having a homozygote or a heterozygote of HLA-A33 or HLA-A 01. That is, the present invention provides immunotherapy for cancers expressing FOXM1 and at least one HLa antigen selected from HLA-A33 and HLA-A 01.
It is another object of the present invention to provide a method of inducing an immune response against cancer in a subject, wherein the method comprises the step of administering to the subject a composition comprising the peptide of the present invention or a polynucleotide encoding the peptide, the APC of the present invention, exosomes presenting the peptide of the present invention, and/or the CTL of the present invention. It is another object of the present invention to provide a method for treating and/or preventing cancer and preventing postoperative recurrence thereof in a subject, wherein the method comprises the step of administering the peptide of the present invention, the polynucleotide encoding the peptide, the APC of the present invention, the exosomes presenting the peptide of the present invention, and/or the CTL of the present invention to the subject.
In addition to the foregoing, other objects and features of the present invention will become more apparent when the following detailed description is read in conjunction with the accompanying drawings and examples. It is to be understood, however, that both the foregoing general description and the following detailed description are exemplary embodiments, but are not restrictive of the invention or other alternative embodiments thereof. In particular, while the invention has been described herein with reference to a number of specific embodiments, it should be understood that the description is illustrative of the invention and is not to be construed as limiting the invention. Various modifications and applications may occur to those skilled in the art without departing from the spirit and scope of the invention as described in the appended claims. Likewise, other objects, features, benefits and advantages of the present invention will become apparent from this summary and certain embodiments described below, and will be apparent to those skilled in the art. Such objects, features, benefits and advantages will be apparent from the above description in connection with the accompanying examples, data, figures and all reasonable inferences drawn therefrom, alone or in conjunction with the references incorporated herein.
Drawings
FIG. 1-1 is composed of photographs (a) to (v) showing the results of an Interferon (IFN) -gamma enzyme-linked immunospot (ELISPOT) assay using peptide-induced cells derived from FOXM 1. In these figures, "+" shows IFN-gamma production for target cells that were impacted with the peptide of interest, and "-" shows IFN-gamma production for target cells that were not impacted with any peptide (negative control). As can be seen by comparison with the negative control, peptide specific IFN-gamma production was observed in the following wells:
using the FOXM1-A33-9-180 (SEQ ID NO: 1) well #6 (a),
Using the hole #3 (b) of FOXM1-A33-9-308 (SEQ ID NO: 2),
Using the FOXM1-A33-9-693 (SEQ ID NO: 3) well #4 (c),
Using the hole #3 (d) of FOXM1-A33-9-516 (SEQ ID NO: 6),
Using the FOXM1-A33-9-146 (SEQ ID NO: 7) well #5 (e),
Using the FOXM1-A33-9-289 (SEQ ID NO: 11) well #6 (f),
Using the FOXM1-A33-9-228 (SEQ ID NO: 12) well #6 (g),
Using the hole #4 (h) of FOXM1-A33-9-502 (SEQ ID NO: 17),
Using the FOXM1-A33-9-321 (SEQ ID NO: 18) well #2 (i),
Using the hole #6 (j) of FOXM1-A33-9-341 (SEQ ID NO: 20),
Using the FOXM1-A33-10-514 (SEQ ID NO: 22) well #8 (k),
Using the FOXM1-A33-10-179 (SEQ ID NO: 24) well #6 (l),
Using the FOXM1-A33-10-501 (SEQ ID NO: 26) well #5 (m),
Using the hole #5 (n) of FOXM1-A33-10-124 (SEQ ID NO: 32),
Using the FOXM1-A33-10-595 (SEQ ID NO: 33) well #3 (o),
Using the FOXM1-A33-10-546 (SEQ ID NO: 36) well #5 (p),
Using the FOXM1-A33-10-391 (SEQ ID NO: 39) hole #6 (q),
Using the FOXM1-A33-10-607 (SEQ ID NO: 41) well #3 (r),
Using the hole # 2(s) of FOXM1-A33-10-265 (SEQ ID NO: 42),
Well #6 (t) using FOXM1-A33-10-4 (SEQ ID NO: 45), and
Hole #5 (u) of FOXM1-A33-10-388 (SEQ ID NO: 46) was used. Cells circled in the photographs showing the response proliferate to establish CTL cell lines. Meanwhile, FOXM1-A33-10-288 (SEQ ID NO: 25) (v) shows an example of typical negative data in which NO peptide-specific IFN-gamma production was observed.
Fig. 1-2 continues to show fig. 1-1.
FIG. 2 is a graph (a) to (d) showing the measurement results of IFN-gamma produced by CTL lines stimulated with FOXM1-A33-9-308 (SEQ ID NO: 2) (a), FOXM1-A33-9-146 (SEQ ID NO: 7) (b), FOXM1-A33-10-391 (SEQ ID NO: 39) (c) or FOXM1-A33-10-265 (SEQ ID NO: 42) (d) using an IFN-gamma enzyme-linked immunosorbent assay (ELISA). These results show that CTL cell lines producing IFN-gamma in a peptide-specific manner were established after induction with each peptide. In the figure, "+" shows IFN-gamma production for CTL lines against target cells impacted with the peptide of interest, and "-" shows IFN-gamma production for CTL lines against target cells not impacted with any peptide. The R/S ratio represents the ratio of the number of cells of the CTL line (responding cells) to the number of cells of the target cells that stimulated them (stimulated cells).
FIG. 3 consists of a series of line graphs (a) to (b) showing IFN-gamma production in CTL clones established by limiting dilution method after induction with FOXM1-A33-9-308 (SEQ ID NO: 2) (a) or FOXM1-A33-9-146 (SEQ ID NO: 7) (b). These results show peptide-specific IFN-gamma production by CTL clones. In the figure, "+" shows IFN-gamma production for CTL clones of target cells that were impacted with the peptide of interest, and "-" shows IFN-gamma production for CTL clones of target cells that were not impacted with any peptide. The R/S ratio represents the ratio of the number of cells of the CTL clone (responding cells) to the number of cells of the target cells (stimulating cells) that stimulated them.
FIG. 4 is a line graph showing IFN-gamma production by CTL clones directed against target cells expressing both FOXM1 and HLA-A 33:03. Target cells into which HLA-A 33:03 or full length FOXM1 gene was introduced were used as negative controls. Induction of established CTL clones using FOXM1-A33-9-308 (SEQ ID NO: 2) showed IFN-gamma production (black diamonds) for COS7 introduced into both FOXM1 and HLA-A 33:03. On the other hand, no significant IFN-gamma production was shown for COS7 cells introduced into one of HLA-A 33:03 (white triangle) and FOXM1 (white circle).
FIG. 5-1 is composed of photographs (a) to (n) showing the results of an IFN-gamma enzyme-linked immunospot (ELISPOT) assay using cells induced with a peptide derived from FOXM 1. In the figure, "+" shows IFN-gamma production for target cells that were impacted with the peptide of interest, and "-" shows IFN-gamma production for target cells that were not impacted with any peptide (negative control).
As can be seen by comparison with the negative control, peptide specific IFN-gamma production was observed in the following wells:
in well #3 using FOXM1-A01-9-233 (SEQ ID NO: 48) (a),
In well #3 using FOXM1-A01-9-539 (SEQ ID NO: 49) (b),
In well #3 using FOXM1-A01-9-631 (SEQ ID NO: 50) (c),
In well #2 using FOXM1-A01-9-231 (SEQ ID NO: 52) (d),
In well #2 using FOXM1-A01-9-663 (SEQ ID NO: 53) (e),
In well #5 (f) using FOXM1-A01-9-494 (SEQ ID NO: 55),
In well #2 (g) using FOXM1-A01-9-341 (SEQ ID NO: 20),
In well #1 (h) using FOXM1-A01-10-566 (SEQ ID NO: 56),
In well #2 using FOXM1-A01-10-263 (SEQ ID NO: 57) (i),
In well #4 (j) using FOXM1-A01-10-308 (SEQ ID NO: 58),
In well #6 (k) using FOXM1-A01-10-232 (SEQ ID NO: 59),
In well #6 (l) and using FOXM1-A01-10-663 (SEQ ID NO: 60)
In well #6 (m) using FOXM1-A01-10-341 (SEQ ID NO: 61).
Cells responding to the display of the picture frame proliferate to establish a CTL line. Meanwhile, FOXM1-A01-10-265 (SEQ ID NO: 42) (n) shows an example of typical negative data in which NO peptide-specific IFN-gamma production was observed.
Fig. 5-2 continues to show fig. 5-1.
FIG. 6 is a line graph showing the results of measuring IFN-gamma produced by CTL lines stimulated with FOXM1-A01-10-566 (SEQ ID NO: 56) by ELISA. The results show that, following induction with peptide, CTL lines were established that produced IFN-gamma in a peptide-specific manner. In the figure, "+" shows IFN-gamma production for CTL lines against target cells impacted with the peptide of interest, and "-" shows IFN-gamma production for CTL lines against target cells not impacted with any peptide. The R/S ratio represents the ratio of the number of cells of the CTL line (responding cells) to the number of cells of the target cells that stimulated them (stimulated cells).
FIG. 7 is a series of line graphs (a) to (b) showing IFN-gamma production in CTL clones established by limiting dilution method after induction with FOXM1-A01-9-233 (SEQ ID NO: 48) or FOXM1-A01-10-566 (SEQ ID NO: 56). These results show peptide-specific IFN-gamma production by CTL clones. In the figure, "+" shows IFN-gamma production for CTL lines against target cells impacted with the peptide of interest, and "-" shows IFN-gamma production for CTL lines against target cells not impacted with any peptide. The R/S ratio represents the ratio of the number of cells of the CTL clone (responding cells) to the number of cells of the target cells (stimulating cells) that stimulated them.
FIG. 8 is a line graph showing IFN-gamma production by CTL clones directed against target cells expressing both FOXM1 and HLA-A 01:01. Target cells into which HLA-A 01:01 or full length FOXM1 gene was introduced were used as negative controls. Induction of established CTL clones using FOXM1-A01-10-566 (SEQ ID NO: 56) showed IFN-gamma production (black diamonds) for COS7 introduced into both FOXM1 and HLA-A. Times.01:01. On the other hand, no significant IFN-gamma production was shown for COS7 cells introduced into one of HLA-A 01:01 (white triangle) and FOXM1 (white circle).
Detailed Description
Description of the embodiments
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. Before describing the materials and methods of the present invention, however, it is to be understood that this invention is not limited to the particular sizes, shapes, dimensions, materials, methodologies, protocols, etc., described herein as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the present description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
I. definition of the definition
The words "a" and "an" as used herein mean "at least one" unless specifically indicated otherwise.
The terms "isolated" and "purified" when used in connection with a substance (e.g., peptide, antibody, polynucleotide, etc.) refer to the substance being substantially free of at least one substance that may be included in a natural source. Thus, an isolated or purified peptide refers to a peptide that is substantially free of another cellular material, such as carbohydrates, lipids, or other contaminating proteins from the cellular or tissue source of the peptide. When chemically synthesizing a peptide, an isolated or purified peptide refers to a peptide that is substantially free of a precursor substance or another chemical substance. The phrase "substantially free of cellular material" includes preparations of peptides in which the peptide is isolated from cellular components of cells from which the peptide was isolated or from which the peptide was recombinantly produced. Thus, peptides that are substantially free of cellular material include peptide preparations that contain less than about 30%,20%,10%, or 5%,3%,2%, or 1% (by dry weight) of other cellular material.
When the peptide is recombinantly produced, the isolated or purified peptide is substantially free of culture medium, and the peptide substantially free of culture medium comprises a peptide preparation comprising less than about 20%, 10%, or 5%, or 3%, 2%, or 1% (by dry weight) of culture medium by volume of the peptide preparation.
Or when chemically synthesizing a peptide, the isolated or purified peptide is substantially free of precursor species or other chemicals, and the peptide substantially free of precursor species or other chemicals contains a peptide preparation comprising less than about 30%, 20%, 10%, 5%, 3%, 2%, or 1% (by dry weight) of the precursor species or other chemicals by volume of the peptide preparation. The isolation or purification of the peptide preparation can be confirmed by, for example, single bands after Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel electrophoresis and coomassie blue staining or such gels. In a preferred embodiment, the peptides and polynucleotides of the invention are isolated or purified.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. In addition to naturally occurring amino acid polymers, these terms also apply to non-naturally occurring amino acid polymers that comprise one or more non-naturally occurring amino acid residues. Non-naturally occurring amino acids include amino acid analogs, amino acid mimics, and the like.
The term "amino acid" as used herein refers to naturally occurring amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code in the cell, as well as those post-translationally modified (e.g., hydroxyproline, gamma-carboxyglutamic acid, O-phosphoserine, etc.). The phrase "amino acid analog" refers to a chemical compound (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium, etc.) that has the same basic chemical structure as a naturally occurring amino acid (alpha carbon, carboxyl, amino and R groups combined with hydrogen) but has a modified R group or modified backbone. The phrase "amino acid mimetic" refers to a compound that has a different structure from a general amino acid but has a similar function to an amino acid. The amino acid may be an L-amino acid or a D-amino acid, and the peptide of the present invention is preferably an L-amino acid polymer.
The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein and refer to a polymer of nucleotides.
The term "composition" as used in this specification is intended to include products comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts. When the composition is a pharmaceutical composition, the term "composition" is intended to include products of the active ingredient and inert ingredient, as well as any product resulting directly or indirectly from the combination, complexation or aggregation of any two or more of the ingredients, from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Thus, the pharmaceutical compositions of the invention include any composition prepared by mixing a compound or cell of the invention with a pharmaceutically or physiologically acceptable carrier. The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used in this specification includes, but is not limited to, liquid or solid fillers, diluents, excipients, solvents and encapsulating materials, and refers to pharmaceutically or physiologically acceptable materials, compositions, substances or mediums.
The term "cancer" refers to a cancer that overexpresses the FOXM1 gene unless otherwise specified, and examples thereof include Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, testicular tumor, and the like, but are not limited thereto. In an exemplary embodiment, a "cancer" is a cancer that expresses FOXM1 and HLA-A33 and/or HLA-A 01.
The terms "cytotoxic T lymphocyte" and "cytotoxic T cell" and "CTL" are used interchangeably herein unless otherwise indicated. Unless specifically stated otherwise, they refer to a subset of T lymphocytes that recognize non-self cells (e.g., tumor/cancer cells, virally infected cells) and induce the death of such cells.
Unless otherwise indicated, the term "HLA-A33" is intended to include subtypes such as HLA-A *33:03,HLa-a* 33:01, and HLA-A * 33:04, of type HLA-A 33.
Unless otherwise indicated, the term "HLA-A01" is intended to include the HLA-A01 forms that include subtypes such as HLA-A *01:01,HLa-a* 01:03, and HLA-A * 01:01:04.
In the context of a subject or patient, the phrase "the HLA antigen of the subject (or patient) is HLA-a33" as used herein means that the subject or patient has homozygous or heterozygous HLA-a33 antigen gene as an MHC (major histocompatibility complex) class I molecule, and that the HLA-a33 antigen is expressed as an HLA antigen in cells of the subject or patient. Similarly, the phrase "the HLA antigen of a subject (or patient) is HLA-A01" as used herein means that the subject or patient has homozygous or heterozygous HLA-A01 antigen gene as an MHC (major histocompatibility complex) class I molecule, and that the HLA-A01 antigen is expressed as an HLA antigen in cells of the subject or patient.
Insofar as the methods and compositions of the invention are useful in the context of "treatment" of cancer, the treatment is considered "effective" when it achieves clinical advantages, e.g., reducing the size, spread or metastatic capacity of cancer in a subject, slowing the progression of cancer, alleviating clinical symptoms of cancer, prolonging survival, inhibiting postoperative recurrence. When the treatment is administered prophylactically, "effective" refers to treatment that delays or prevents the formation of cancer, or prevents or alleviates clinical symptoms of cancer. Effectiveness is determined relative to any known method for diagnosing or treating a particular tumor type.
Whenever the methods and compositions of the present invention are used in the context of "prevention (guard)" of cancer, the term "guard (guard)" herein includes any effort to reduce the burden of mortality or morbidity associated with cancer. Prevention can be performed at "primary, secondary and tertiary prevention (guard) levels". Although primary prevention (prophylaxis) avoids the development of the disease, prevention (prophylaxis) at both secondary and tertiary levels includes preventing (prophylaxis) disease progression and appearance of symptoms, as well as aims to reduce the adverse effects of existing diseases by restoring function and reducing disease-related complications. Or prophylaxis (prevention) may include a broad prophylactic treatment that reduces the severity of a particular condition, e.g., aims to reduce tumor growth and metastasis.
In the context of the present invention, the treatment and/or prevention (prevention) of cancer and/or the prevention (prevention) of postoperative recurrence thereof includes any event, such as inhibition of cancer cell proliferation, tumor regression or regression, induction of remission and inhibition of cancer progression, tumor regression, and reduction or inhibition of metastasis, inhibition of postoperative recurrence of cancer and prolongation of survival. Effective treatment and/or prevention of cancer reduces mortality, improves prognosis of individuals with cancer, reduces blood levels of tumor markers, and reduces detectable symptoms associated with cancer. For example, alleviation or amelioration of symptoms constitutes an effective treatment and/or prevention (prophylaxis) and includes patients in which symptoms are stabilized or alleviated by 10%,20%,30% or more.
In the context of the present invention, the term "antibody" refers to immunoglobulins and fragments thereof that specifically react with a specified protein or peptide thereof. Antibodies may include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments. Furthermore, "antibody" is used herein in the broadest sense and specifically covers whole monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from two or more whole antibodies, and antibody fragments so long as they exhibit the desired biological activity. An "antibody" may be an antibody of all classes (e.g., igA, igD, igE, igG and IgM).
Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
II peptides
HLA-A33 is an allele common to Asians and HLA-A01 is an allele common to Cao et al, hum Immunol 2001;62 (9): 1009-30). Thus, for a large population of asians or caucasians, an effective method of treating FOXM 1-expressing cancers may be provided by providing FOXM 1-derived CTL-inducing peptides restricted to HLA-A33 or HLA-A 01. The present invention thus provides FOXM 1-derived peptides capable of inducing CTLs in a HLA-A 33-or HLA-A 01-restricted manner.
The peptides of the invention are FOXM 1-derived peptides capable of inducing CTLs in a HLA-A 33-or HLA-A 01-restricted manner. Peptides capable of inducing CTLs in a HLA-A33 restricted manner include peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 2, 3,6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46. Peptides capable of inducing CTL in a HLA-A01 restricted manner include peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61.
CTLs with cytotoxic activity specific for these peptides can be established by in vitro stimulation of T cells by Dendritic Cells (DCs) impacted (pulsed) with these peptides. The established CTLs showed specific cytotoxic activity against target cells impacted with each peptide.
FOXM1 gene is overexpressed in cancer cells, e.g., in cancer cells in Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, testicular tumor, etc., but not in most normal organs. It is therefore an excellent target for immunotherapy. Thus, the peptides of the invention may be suitably used in cancer immunotherapy. Preferred peptides are nonapeptides (peptides consisting of 9 amino acid residues) or decapeptides (peptides consisting of 10 amino acid residues), and more preferred are peptides ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58,59、60 and 61 selected from the group consisting of the following amino acid sequences. For example, peptides having the amino acid sequence of SEQ ID NO. 2 are suitable for inducing CTLs exhibiting specific cytotoxic activity against cells expressing HLA-A33 and FOXM1, and may be suitable for cancer immunotherapy in patients positive for HLA-A 33. In a more preferred embodiment, the peptide of the invention is a peptide consisting of the amino acid sequence of SEQ ID NO. 2. In addition, for example, a peptide having the amino acid sequence of SEQ ID NO:56 is suitable for inducing CTLs exhibiting specific cytotoxic activity against cells expressing HLA-A01 and FOXM1, and can be suitably used for cancer immunotherapy against HLA-A 01-positive patients. In a more preferred embodiment, the peptide of the invention is a peptide consisting of the amino acid sequence of SEQ ID NO. 56.
For the peptide of the present invention, additional amino acid residues may be prepared to add (adjoin) the amino acid sequence of the peptide of the present invention, as long as the resulting peptide retains the CTL inducibility of the original peptide. The additional amino acid residues may be composed of any type of amino acid as long as they do not impair the CTL inducibility of the original peptide. Thus, the peptide of the present invention comprises a peptide having CTL inducibility, which comprises amino acid sequences ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61 selected from the group consisting of. Such peptides are, for example, less than about 40 amino acids, in many cases less than about 20 amino acids, and typically less than about 15 amino acids. Thus, if the original peptide is a nonapeptide, the peptide of the present invention comprises a 10 amino acid long or 11-40 amino acid long peptide produced by attaching additional amino acids to the peptide. Furthermore, if the original peptide is a decapeptide, the peptide of the present invention comprises a peptide 11-40 amino acids long. Such peptides may be, for example, 11-20 amino acid long peptides or 11-15 amino acid long peptides. A preferred example of an additional amino acid residue is an amino acid residue (e.g., SEQ ID NO:67, 69, 71, 73 or 75) that abuts the amino acid sequence of the peptide of the present invention in the full-length amino acid sequence of FOXM 1. Thus, the peptides of the present invention include peptides ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61 comprising an amino acid sequence selected from the group consisting of, and wherein the peptides are peptide fragments of FOXM1 and have CTL inducibility.
In general, modification of one, two or more amino acids in certain peptides will not affect the function of the peptide or in some cases will even enhance the desired function of the original protein. In fact, modified peptides (i.e., peptides composed of amino acid sequences in which one, two, or several amino acid residues are modified (i.e., substituted, added, deleted, and/or inserted) as compared to the original reference sequence) are known to retain the biological activity of the original peptide (Mark et al .,Proc Natl Acad Sci USA 1984,81:5662-6;Zoller and Smith,Nucleic Acids Res 1982,10:6487-500;Dalbadie-McFarland et al, proc NATL ACAD SCI USA 1982, 79:6409-13). Thus, in one embodiment, the peptide of the present invention may be a peptide comprising an amino acid sequence in which one, two or several amino acids are substituted, deleted, inserted and/or added to an amino acid sequence selected from SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61, and having CTL inducibility.
Those skilled in the art will recognize that individual substitutions to an amino acid sequence (which alter a single amino acid or a small percentage of amino acids) tend to result in retention of the properties of the original amino acid side chains. Which is commonly referred to as a "conservative substitution" or "conservative modification", and modifications to a protein by which a "conservative substitution" or "conservative modification" may result in a modified protein that has a similar function as the original protein. Conservative substitutions providing functionally similar amino acids are well known in the art. Examples of functionally similar amino acid side chain characteristics include, for example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having common functional groups or characteristics of aliphatic side chains (G, A, V, L, I, P), hydroxyl-containing side chains (S, T, Y), sulfur atom-containing side chains (C, M), carboxylic acid and amide-containing side chains (D, N, E, Q), base-containing side chains (R, K, H), and aromatic-containing side chains (H, F, Y, W). In addition, the following eight groups each contain amino acids that are recognized in the art as conservative substitutions for one another:
1) Alanine (a), glycine (G);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (N), glutamine (Q);
4) Arginine (R), lysine (K);
5) Isoleucine (I), leucine (L), methionine (M), valine (V);
6) Phenylalanine (F), tyrosine (Y), tryptophan (W);
7) Serine (S), threonine (T), and
8) Cysteine (C), methionine (M) (see, e.g., cright on, proteins 1984).
Such conservatively modified peptides are also encompassed by the peptides of the invention. However, the peptide of the present invention is not limited thereto, and may include non-conservative modifications as long as the peptide retains CTL inducibility of the original peptide. In addition, modified peptides do not exclude CTL-inducible peptides derived from polymorphic variants, interspecies homologs, and alleles of FOXM 1.
A few (e.g., 1,2, or several) or a small percentage of amino acids may be modified (i.e., substituted, deleted, inserted, and/or added) so long as the peptide retains the CTL inducibility of the original peptide. Here, the term "several" means 5 or less amino acids, such as 4 or 3 or less. The percentage of modified amino acids is preferably 20% or less, more preferably 15% or less, still more preferably 10% or less, or 1 to 5%.
When used in the context of immunotherapy, the peptides of the invention should be presented on the surface of cells or exosomes, preferably as complexes with HLA antigens. Thus, it is preferred that the peptides of the invention possess high binding affinity for HLA antigens. To this end, the peptides may be modified by substitution, deletion, insertion and/or addition of amino acid residues to produce modified peptides with improved binding affinity. Since the sequence rules of peptides displayed by binding to HLA antigens are known (Falk, et al, immunogenetics 1994 40 232-41; chujoh, et al, tissue Antigens 1998:52:501-9; takiuchi, et al, tissue Antigens 2000:55:296-302), modifications based on such rules can be introduced into the peptides of the invention.
For example, in peptides having binding affinity for HLA class I molecules, the second amino acid from the N-terminus and the C-terminal amino acid are typically anchor residues involved in binding to HLA class I (RAMMENSEE HG, et al, immunogenetics.1995;41 (4): 178-228). For example, in HLA-A33, for the second amino acids phenylalanine, tyrosine, alanine, isoleucine, leucine and valine from the N-terminus, and for the arginine and lysine of the C-terminal amino acid, it is known as an anchor residue with high binding affinity for HLA-A33 (false, et al Immunogenetics 1994,40:232-41;Takiguchi,et al, tissue Antigens 2000, 55:296-302).
In addition, in HLA-A33, the first amino acid residue from the N-terminus is known to function as an anchor residue, and aspartic acid and glutamic acid are known to be preferred as the first amino acids from the N-terminus (Falk, et al, immunogenetics 1994,40:232-41;Takiguchi,et al, tissue Antigens 2000:55:296-302). Thus, in order to maintain or enhance HLA-A33 binding affinity, it may be desirable to replace a first amino acid from the N-terminus with aspartic acid or glutamic acid, and a second amino acid from the N-terminus with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine, and/or a C-terminal amino acid with arginine or lysine.
Thus, peptides comprising the following amino acid sequences, wherein in the amino acid sequences selected from the group consisting of SEQ ID NOs 1,2, 3, 6,7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46, the first amino acid is substituted with aspartic acid or glutamic acid from the N-terminus, the second amino acid is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine from the N-terminus, and/or the C-terminal amino acid is substituted with arginine or lysine, are included in the peptides of the present invention.
In a preferred embodiment, the peptide of the present invention may be a peptide consisting of an amino acid sequence having CTL inducibility, wherein in the amino acid sequence selected from the group consisting of SEQ ID NOs 1,2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46, a first amino acid is substituted with aspartic acid or glutamic acid from the N-terminus, a second amino acid is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine from the N-terminus, and/or a C-terminal amino acid is substituted with arginine or lysine.
That is, the peptide of the present invention includes a peptide having a CTL-inducing ability comprising an amino acid sequence selected from the group consisting of 1,2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 in the amino acid sequence of SEQ ID NOs, having one or more substituted amino acid sequences selected from the group consisting of (a) to (c) below:
(a) The first amino acid from the N-terminus is substituted with aspartic acid or glutamic acid;
(b) The second amino acid from the N-terminus being substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine, and
(C) The C-terminal amino acid is substituted with arginine or lysine.
In a preferred embodiment, the peptide of the present invention may be a peptide consisting of an amino acid sequence having CTL inducibility, wherein one or more substitutions selected from the group consisting of (a) to (c) above are introduced into the amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 6, 7, 11,12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46. In the present invention, the preferred number of substitutions is 1,2 or 3 substitutions selected from the above (a) to (c).
Furthermore, the peptide of the present invention may be a peptide having a CTL-inducing ability comprising an amino acid sequence in which the second amino acid is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine from the N-terminus and/or the C-terminal amino acid is substituted with arginine or lysine in an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2,3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46. Preferably, the peptide of the present invention may be a peptide consisting of an amino acid sequence having CTL inducibility, wherein in the amino acid sequence selected from the group consisting of SEQ ID NOs 1,2,3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46, the second amino acid is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine from the N-terminus, and/or the C-terminal amino acid is substituted with arginine or lysine. That is, the peptide of the present invention may be a peptide having CTL inducibility, which has one or more substituted amino acid sequences selected from the group consisting of the following (a) and (b) in the amino acid sequences selected from the group consisting of SEQ ID NOs:1, 2,3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46:
(a) The second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine;
(b) The C-terminal amino acid is substituted with arginine or lysine.
In a preferred embodiment, the peptide of the present invention may be a peptide having CTL inducibility, which consists of an amino acid sequence having one or more substitutions selected from the group consisting of (a) and (b) above in the amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46. In a more preferred embodiment, the second amino acid from the N-terminus is substituted with phenylalanine or tyrosine.
In HLA-A01, aspartic acid and glutamic acid for the third amino acid from the N-terminus, and tyrosine for the C-terminal amino acid are known to be anchor residues with high binding affinity for HLA-A 01. Furthermore, it is known that HLA-A01 has an auxiliary anchor residue at position 2 from the N-terminus, and threonine and serine are known to be preferable as the second amino acid from the N-terminus (Kubo,R.T Journal of Immunology 1994,152:3913;Gambacorti-Passerini,C.Clinical Cancer Research 1997,3:675-83;Falk,K.Immunogenetics 1994,40:238-41).
Thus, in order to maintain or enhance HLA-A01 binding affinity, it may be desirable to replace the third amino acid from the N-terminus with aspartic acid or glutamic acid, and/or to replace the C-terminal amino acid with tyrosine. Another possibility is to replace the second amino acid from the N-terminus with threonine or serine. Thus, a peptide comprising the following amino acid sequence, wherein in the amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61, the second amino acid from the N-terminus is substituted with threonine or serine, the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, and/or the C-terminal amino acid is substituted with tyrosine, having CTL-inducing ability is included in the peptide of the present invention.
In a preferred embodiment, the peptide of the present invention may be a peptide consisting of an amino acid sequence having CTL inducibility, wherein in the amino acid sequence selected from the group consisting of SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61, the second amino acid from the N-terminus is substituted with threonine or serine, the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, and/or the C-terminal amino acid is substituted with tyrosine.
That is, the peptide of the present invention includes a peptide having a base acid sequence having CTL inducibility, which is contained in an amino acid sequence selected from the group consisting of SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61, having one or more substituted amino acid sequences selected from the group consisting of (a) to (c) below:
(a) The second amino acid from the N-terminus is substituted with threonine or serine;
(b) The third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid;
(c) The C-terminal amino acid is substituted with tyrosine.
In a preferred embodiment, the peptide of the present invention may be a peptide consisting of an amino acid sequence having CTL inducibility, wherein the amino acid sequence has one or more substitutions selected from the group consisting of (a) to (c) above in an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61. In the present invention, the preferred number of substitutions is 1,2 or 3 substitutions selected from the above (a) to (c).
Furthermore, the peptide of the present invention may be a peptide having a CTL-inducing ability comprising an amino acid sequence in which the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid and/or the C-terminal amino acid is substituted with tyrosine in the amino acid sequence selected from SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61. Preferably, the peptide of the present invention may be a peptide consisting of an amino acid sequence having CTL inducibility, wherein in the amino acid sequences of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61, the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, and/or the C-terminal amino acid is substituted with tyrosine. That is, the peptide of the present invention includes a peptide having a CTL inducibility comprising an amino acid sequence having one or more substitutions selected from the group consisting of the following (a) and (b) in an amino acid sequence selected from the group consisting of SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61:
(a) A third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, and
(B) The C-terminal amino acid is substituted with tyrosine.
In a preferred embodiment, the peptide of the present invention may be a peptide consisting of an amino acid sequence having CTL inducibility, wherein the amino acid sequence has one or more substitutions selected from the group consisting of (a) to (b) above in an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61.
Not only amino acid substitutions can be introduced at the anchor site, but also at the potential T Cell Receptor (TCR) recognition site of the peptide. Several studies have demonstrated that peptides with amino acid substitutions can be equivalent or better than the original functions, such as CAP1, p53 (264-272)、Her-2/neu(369-377), or gp100 (209-217) (Zaremba et al Cancer Res.57,4570-4577,1997, T.K. Hoffmann et al J Immunol. (2002); 168 (3): 1338-47, S.O. Dionne et al Cancer Immunol. (2003) 52:199-206, and S.O. Dionne et al Cancer Immunology, immunotherapy (2004) 53, 307-314).
The present invention also contemplates that 1,2 or several amino acids may also be added to the N-terminus and/or C-terminus of the peptides of the present invention (e.g., peptides ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61 consisting of amino acid sequences selected from the group consisting of. More specifically, the present invention provides peptides consisting of amino acids in which one, two or several amino acids are added to either or both of the N-terminal and C-terminal ends of the amino acid sequences shown in the respective SEQ ID NOs. Such modified peptides that retain CTL inducibility are also included in the present invention. For example, when a peptide is contacted with an APC (wherein two or more amino acids are added to the N-terminus and/or C-terminus of a peptide of the amino acid sequence consisting of SEQ ID NOs:2 or 56), it is incorporated into the APC and processed into a peptide consisting of the amino acid sequence of SEQ ID NOs:2 or 56. It can then induce CTLs by presenting on the cell surface of APCs via the antigen presentation pathway. More specifically, the peptide of the present invention may be a peptide in which one, two or several amino acids are added to either or both of the N-terminus and the C-terminus.
Furthermore, in another embodiment of the invention, peptides are provided which consist of an amino acid sequence comprising one, two or several amino acid substitutions in the amino acid sequence shown in the respective SEQ ID NOs, and wherein one, two or several amino acids are added to one or both of the N-terminal and C-terminal ends of these substituted amino acid sequences.
When the peptide of the present invention comprises an amino acid substitution, the desired substitution position may be, for example, a first position from the N-terminus, a second position from the N-terminus, and one, two or three positions from the C-terminus in the amino acid sequence shown by SEQ ID NOs:1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46, or a second position from the N-terminus, two or three positions from the C-terminus in the amino acid sequence shown by SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61.
However, when the amino acid sequence of the peptide is identical to a part of the amino acid sequence of an endogenous or exogenous protein having a different function, side effects such as autoimmune diseases and/or allergic symptoms against a specific substance can be induced. Therefore, homology searches are preferably performed using available databases to avoid situations where the amino acid sequence of a peptide matches the amino acid sequence of another protein. When it is clear from the homology search that there is no peptide differing by only 1 or 2 amino acids from the target peptide, the target peptide may be modified to increase its binding affinity to HLA antigen and/or to increase its CTL inducibility without the risk of such side effects.
Peptides in which one, two or several amino acids of the peptides of the present invention are modified are predicted to retain the CTL-inducing ability of the original peptide, however, it is preferable to verify the CTL-inducing ability of the modified peptide. Herein, "peptide having CTL inducibility (CTL inducibility)" refers to a peptide that induces CTLs by APC stimulated with the peptide.
"CTL induction" includes induction of differentiation into CTL, induction of CTL activation, induction of CTL proliferation, induction of cytotoxic activity of CTL, induction of CTL-mediated lysis of target cells, and induction of increased IFN-gamma production of CTL.
CTL inducibility can be confirmed by stimulating APCs (e.g., B lymphocytes, macrophages or dendritic cells) expressing the HLA antigen of interest with a peptide and mixing it with CD8 positive cells, and then measuring IFN-gamma released by the CTL against the target cells. For APCs, dendritic cells derived from human peripheral blood mononuclear cells can be preferably used. As a reaction system, transgenic animals that have been made to express HLA antigens can be used. Alternatively, target cells may be labeled with 51 Cr or such radioactivity, for example, and the cytotoxic activity of the peptide-induced CTLs may be calculated from the radioactivity released from the target cells. Alternatively, in the presence of peptide-stimulated APCs, it is possible to evaluate CTL-inducing ability by measuring IFN-gamma generated and released by CTLs and visualizing the inhibitory region on the medium using an anti-IFN-gamma monoclonal antibody.
In addition to the modifications described above, the peptides of the invention may be linked to other peptides as long as the resulting linked peptides retain CTL inducibility. Examples of suitable peptides linked to the peptides of the invention include CTL-inducing peptides derived from other TAAs. Furthermore, the peptides of the invention may also be linked to each other. Suitable linkers for linking peptides are known in the art, and linkers such as AAY(P.M.Daftarian et al.,J Trans Med 2007,5:26)、AAA、NKRK(SEQ ID NO:62)(R.P.M.Sutmuller et al.,J Immunol.2000,165:7308-15), or K (S.Ota et al, can Res.62,1471-6;K.S.Kawamura et al, J Immunol.2002, 168:5709-15) may be used, for example. The peptides may be linked (e.g., in a chain, repeat, etc.) in various arrangements, and three or more peptides may also be linked.
The peptides of the invention may also be linked to other substances as long as the resulting linked peptide retains CTL inducibility. Examples of suitable materials for attachment to the peptides of the invention include, for example, peptides, lipids, sugars or sugar chains, acetyl groups, and natural or synthetic polymers. The peptides of the present invention may be modified by glycosylation, side chain oxidation, or phosphorylation, etc., as long as their CTL-inducing ability is not impaired. Modifications of this type may also be implemented to confer additional functions (e.g., targeting and delivery functions) or to stabilize the peptide.
For example, it is known in the art to introduce D-amino acids, amino acid mimics or unnatural amino acids in order to improve the in vivo stability of the peptides, and this concept is also applicable to the peptides of the invention. The stability of a peptide can be determined in a number of ways. For example, stability can be tested by using peptidases as well as various biological mediators such as human plasma and serum (see, e.g., verhoef et al, eur J Drug Metab Pharmacokin1986, 11:291-302).
Furthermore, as described above, among the above-described modified peptides substituted, deleted, inserted and/or added with 1,2 or several amino acid residues, those having the same or higher activity than the original peptide can be selected or selected. Thus, the present invention also provides methods for screening or selecting modified peptides having the same or higher activity as compared to the original peptide. In particular, the present invention provides a method for screening peptides having CTL inducibility, wherein said method comprises the steps of:
(a) Generating a candidate sequence consisting of an amino acid sequence in which one, two or several amino acid residues are substituted, deleted, inserted and/or added to an original amino acid sequence, wherein the original amino acid sequence consists of an amino acid sequence selected from SEQ ID Nos：1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61;
(b) Selecting a candidate sequence from the candidate sequences generated in (a) that has no significant homology (or sequence homology) to the peptide of any known human gene product other than FOXM 1;
(c) Contacting a peptide consisting of the candidate sequence selected in (b) with an APC;
(d) Contacting the APC of (c) with a CD 8-positive T cell, and
(E) Peptides having CTL inducibility equal to or higher than peptides consisting of the original amino acid sequence are selected.
The peptides of the invention are also described herein as "FOXM1 peptides".
III preparation of the peptides of the invention
The peptides of the invention may be prepared using known techniques. For example, recombinant DNA techniques or chemical synthesis can be used to prepare the peptides of the invention. The peptides of the invention may be synthesized independently, or as longer polypeptides comprising two or more peptides. The peptides of the invention may be isolated from the host cell or synthetic reaction products after they are produced in the host cell using recombinant DNA techniques or chemically synthesized. That is, the peptides of the invention may be purified or isolated so that they are substantially free of other host cell proteins and fragments thereof, or any other chemicals.
The peptides of the invention may contain modifications, such as glycosylation, side chain oxidation, or phosphorylation, etc., provided that the modifications do not disrupt the biological activity of the original peptide. Other exemplary modifications include the incorporation of one or more D-amino acids or other available amino acid mimics, for example, to increase the serum half-life of the peptide.
The peptides of the invention can be obtained by chemical synthesis, based on the selected amino acid sequence. Examples of conventional peptide synthesis methods that may be suitable for synthesis include the methods described in the following documents:
(i)Peptide Synthesis,Interscience,New York,1966;
(ii)The Proteins,Vol.2,Academic Press,New York,1976;
(iii)“Peptide Synthesis”(in Japanese),Maruzen Co.,1975;
(iv)“Basics and Experiment of Peptide Synthesis”(in Japanese),Maruzen Co.,1985;
(v)“Development of Pharmaceuticals”(in Japanese),Continued Vol.14(peptide synthesis),Hirokawa,1991;
(vi) WO99/67288, and
(vii)Barany G.&Merrifield R.B.,Peptides Vol.2,Solid Phase Peptide Synthesis,Academic Press,New York,1980,100-118.
Alternatively, any known method of producing genetically engineered peptides may be used to obtain the peptides of the invention (e.g., morrison J, J Bacteriology 1977,132:349-51; clark-Curtiss & Curtiss, methods in Enzymology (eds. Wu et al) 1983, 101:347-62). For example, first, a suitable vector comprising a polynucleotide encoding the peptide of interest in an expressible form (e.g., downstream of regulatory sequences corresponding to promoter sequences) is prepared and transformed into a suitable host cell. The invention also provides such vectors and host cells. The host cells are then cultured to produce the peptide of interest. The peptides of the invention can also be produced in vitro using an in vitro translation system.
IV. Polynucleotide
The invention also provides polynucleotides encoding any of the peptides of the invention. These include polynucleotides derived from the naturally occurring FOXM1 gene (e.g., genBank accession number NM_202003(SEQ ID NO:52)、NM_001243088(SEQ ID NO:54)、NM_001243089(SEQ ID NO:56)、NM_021953(SEQ ID NO:58) or NM202002 (SEQ ID NO: 60)) and having their conservatively modified nucleotide sequences. As used herein, the phrase "conservatively modified nucleotide sequence" refers to a sequence that encodes the same or substantially the same amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG, and GCU both encode the amino acid alanine. Thus, at any position specified by a codon as alanine, the codon can be changed to any corresponding said codon without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Each nucleic acid sequence encoding a peptide herein also describes each possible silent variation of that nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid which encodes a peptide is implicitly described in each disclosed sequence.
The polynucleotides of the present invention may be composed of DNA, RNA, and derivatives thereof. DNA is suitably composed of bases such as A, T, C and G, and in RNA T is replaced by U.
The polynucleotides of the invention may encode a plurality of the peptides of the invention, with or without an intervening amino acid sequence (INTERVENING AMINO ACID SEQUENCE). For example, the intervening amino acid sequence may provide a cleavage site (e.g., an enzyme recognition sequence) for a polynucleotide or translated peptide. Furthermore, the polynucleotide may include any additional sequences encoding the coding sequences of the peptides of the invention. For example, the polynucleotide may be a recombinant polynucleotide comprising regulatory sequences required for expression of the peptide, or may be an expression vector (e.g., a plasmid) having a marker gene or the like. In general, such recombinant polynucleotides can be prepared by manipulating the polynucleotides by conventional recombinant techniques, such as by using polymerases and endonucleases.
Both recombinant and chemical synthesis techniques can be used to generate polynucleotides of the invention. For example, polynucleotides of the invention may be produced by insertion into a suitable vector that is capable of expression upon transfection into competent cells. Alternatively, the polynucleotides may be amplified using PCR techniques or expressed in a suitable host (see, e.g., sambrook et al Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory, new York, 1989). Alternatively, polynucleotides can be synthesized using solid phase techniques, such as those described in Beaucage SL & Iyer RP, tetrahedron 1992,48:2223-311; matthes et al, EMBO J1984, 3:801-5. The ligation products of several peptides obtainable in this way can be purified as desired and administered in this ligation format. In this case, the linked peptides are processed to produce obstetrically antigen-presenting peptides and elicit CTL-inducing activity of each peptide. Thus, when peptides are linked, combinations of peptides with the same HLA limitations are preferred. Alternatively, the peptides may be administered as a mixture of individual peptides by cleavage of the linking moiety.
V. exosomes (exosomes)
The invention further provides intracellular vesicles called exosomes which present on their surface complexes formed between the peptides of the invention and HLA antigens. Exosomes may be prepared by the methods described in detail, for example, in JPH11-510507 and WO99/03499, and may be prepared with APCs obtained from patients as therapeutic and/or prophylactic (preventative) subjects. The exosomes of the invention may be vaccinated in a similar manner as the peptides of the invention.
The type of HLA antigen contained in the above complex must be matched to the type of subject in need of treatment and/or prevention (precaution). For example, HLA-A33 (e.g., HLA-A *: 03) is an allele that is widely and ubiquitously found in Asian people, and this HLA antigen type is considered suitable for treatment in Asian patients. In addition, HLA-A01 (e.g., HLA-A 01: 01) is an allele that is widely and ubiquitously found in caucasian populations, and these HLa antigen types are considered suitable for treatment in caucasian patients. Typically, clinically, by studying in advance the HLA antigen type of a patient in need of treatment, an appropriate peptide can be selected which has a high level of binding affinity to a specific HLA antigen, or CTL inducibility by antigen presentation mediated by the specific HLA antigen.
The exosomes of the present invention present on their surface complexes of the peptides of the present invention with HLA-A33 or HLA-A 01. When HLA forming a complex with the peptide of the present invention is HLA-A33, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46, and more preferably a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46, or a modified peptide thereof. Furthermore, when HLA forming a complex with the peptide of the present invention is HLA-A01, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61, and more preferably a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61, or a modified peptide thereof.
VI Antigen Presenting Cell (APC)
The invention also provides APCs that present on their surface complexes formed between HLA antigens and the peptides of the invention. Alternatively, the present invention provides APCs having on their surfaces a complex formed between an HLA antigen and a peptide of the present invention. The APCs of the present invention may be isolated APCs. When used in the context of a cell (APC, CTL, etc.), the term "isolated" means that the cell is separated from another type of cell. The APC of the present invention may be APC induced from APC derived from a patient to be subjected to treatment and/or prevention (precaution), and may be administered as a vaccine itself or in combination with other drugs (including the peptide of the present invention, exosomes or CTLs).
The APC of the present invention is not limited to a specific kind of cells, and may be a cell that inhibits antigen presenting proteinaceous on its cell surface to be recognized by lymphocytes such as Dendritic Cells (DCs), langerhans cells, macrophages, B cells, and activated T cells. Since DC is a representative APC having the strongest CTL-inducing activity among APCs, it is preferable to use DC as the APC of the present invention. In the present invention, preferred DCs are isolated DCs derived from humans. Furthermore, the APCs of the present invention may also be a mixture of a plurality of types of cells having an antigen presenting function, and may be a mixture of APCs, each of which presents a different type of the peptides of the present invention.
For example, APCs of the invention can be obtained by isolating DCs from peripheral blood mononuclear cells and then stimulating them with the peptides of the invention in vitro. When the peptide of the present invention is administered to a subject, APCs presenting the peptide of the present invention are induced in the body of the subject. Thus, after administration of the peptide of the invention to a subject, the APC of the invention can be obtained by collecting the APC from the subject. Alternatively, the APCs of the invention may be obtained by contacting APCs collected from a subject with the peptide of the invention.
In order to induce an immune response in a subject against FOXM 1-expressing cancer cells, APCs of the invention may be administered to a subject either by themselves or in combination with other drugs (including peptides, exosomes, or CTLs of the invention). For example, ex vivo administration may include the steps of:
(a) Collecting APCs from a first subject;
(b) Contacting the APC of step (a) with a peptide of the invention, and
(C) Administering the APC of step (b) to a second subject.
The first subject and the second subject may be the same individual, or may be different individuals. When the first subject and the second subject are different individuals, it is preferable that the HLA of the first subject and the second subject are of the same type. The APC obtained from step (b) above may be a vaccine for cancer treatment and/or prevention (prophylaxis).
APCs of the invention obtained as described above have CTL inducibility. The term "CTL inducibility" in the context of APCs refers to the ability of APCs to induce CTLs when contacted with CD8 positive cells. In addition, "CTL-inducing ability (CTL-inducing ability)" includes an ability of APC to induce CTL activation, an ability of APC to induce CTL proliferation, an ability of APC to promote CTL-mediated lysis of target cells, and an ability of APC to enhance CTL-mediated IFN-gamma production. The CTLs induced by APCs of the present invention are CTLs specific for FOXM1 and exhibit specific cytotoxic activity against FOXM 1-expressing cells.
In addition to the methods described above, APCs of the present invention can be prepared by introducing a polynucleotide encoding a peptide of the present invention into APCs in vitro. The polynucleotide to be introduced may be in the form of DNA or RNA. The method of introduction is not particularly limited, and examples thereof include various methods conventionally practiced in the art, such as liposome transfection, electroporation, and calcium phosphate method. More specifically, the methods described in CANCER RES 1996,56:5672-7;J Immunol 1998,161:5607-13;J Exp Med 1996,184:465-72, and JP2000-509281 can be used. By introducing a polynucleotide encoding a peptide of the present invention into an APC, the polynucleotide is transcribed and translated in a cell, and then the resulting peptide is processed by MHC class I and presented on the cell surface of the APC by a presentation pathway.
In a preferred embodiment, the APC of the present invention presents on its cell surface a complex of the peptide of the present invention and HLA-A11 (more preferably HLA-A * 33:03) or HLA-A01 (more preferably HLA-A * 01:01). When HLA forming a complex with the peptide of the present invention is HLA-A33, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1,2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46, and more preferably a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 1,2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46. When HLA forming a complex with the peptide of the present invention is HLA-A01, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61, and more preferably a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61.
The APC of the present invention is preferably an APC induced by a method comprising the steps of (a) or (b) below:
(a) Contacting an APC with a peptide of the invention, said APC expressing at least one HLa selected from HLA-A33 (more preferably HLA-A 33:03) and HLA-A01 (more preferably HLA-A 01:01), or
(B) The polynucleotide encoding the peptide of the invention is introduced into an APC which expresses at least one HLa selected from HLA-A33 (more preferably HLA-A 33: 03) and HLA-A01 (more preferably HLA-A 01: 01).
The peptide of the present invention which is contacted with the APC expressing HLA-A33 is preferably a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 2, 3,6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46, and more preferably a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 2, 3,6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46.
The peptide of the present invention which is contacted with the APC expressing HLA-A01 is preferably a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61, and more preferably a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61.
The present invention provides the use of the peptide of the present invention for preparing a pharmaceutical composition for inducing APC having CTL inducibility. In addition, the present invention provides a method or process for preparing a pharmaceutical composition for inducing APC having CTL inducibility. Further, the present invention provides the peptide of the present invention for inducing APC having CTL inducibility.
VII Cytotoxic T Lymphocytes (CTL)
CTLs induced by the peptides of the invention can be used as vaccines in a similar manner to the peptides of the invention for enhancing immune responses in vivo targeting FOXM1 expressing cancer cells. The present invention thus provides CTLs which are induced or activated by the peptides of the present invention. The CTLs of the present invention are CTLs that target the peptides of the present invention and are capable of binding to complexes of the peptides of the present invention and HLA antigens. Binding of CTLs to the complex is mediated via T Cell Receptors (TCRs) present on the cell surface of the CTLs. The CTL of the present invention may be an isolated CTL. Preferred CTLs are isolated human CTLs. The CTLs of the invention can also be mixtures of CTLs, each targeting a different type of peptide of the invention.
The CTLs of the invention can be obtained from (1) administering the peptides of the invention to a subject, (2) stimulating in vitro APC and CD8 positive T cells derived from the subject, or Peripheral Blood Mononuclear Cells (PBMCs), (3) contacting in vitro CD8 positive T cells or PBMCs with APC or exosomes presenting complexes of HLA antigens and the peptides of the invention on their surfaces, or (4) introducing into CD8 positive T cells a vector comprising a polynucleotide encoding each T Cell Receptor (TCR) subunit capable of binding the peptides of the invention presented by HLA antigens on the cell surfaces. Exosomes and APCs for use in the methods of (2) or (3) above can be prepared by the methods described in the "v. Exosomes" and "vi. Antigen Presenting Cells (APCs)" sections, respectively, and details of the method of (4) above will be described in the "viii. T Cell Receptor (TCR)" section.
The CTLs of the present invention can be administered to a patient undergoing treatment and/or prevention (prophylaxis) alone or in combination with other drugs (including the peptides, APCs, or exosomes of the present invention) for the purpose of modulating effects. Further, the CTLs of the present invention can be CTLs induced from CD 8-positive T cells derived from a patient undergoing administration of CTLs. The CTLs of the present invention specifically act on target cells presenting the peptides of the present invention, e.g., the same peptides used to induce the CTLs of the present invention. The target cell may be a cell that endogenously expresses FOXM1, such as a cancer cell, or a cell transfected with FOXM1 gene. Cells presenting the peptides of the invention on the cell surface as a result of stimulation by the peptides can also be targets for attack by CTLs of the invention. The CTL-targeted cells of the invention are preferably cells positive for at least one of HLA-A33 (more preferably HLA-A x 33:03) and HLA-A01 (more preferably HLA-A x 01:01).
In a preferred embodiment, CTLs of the invention specifically target cells expressing FOXM1 and HLa selected from at least one of HLA-A33 (more preferably HLA-A 33:03) and HLA-A01 (more preferably HLA-A 01:01). In the present invention, the cells targeted by CTL may be homozygous or heterozygous cells having either of the alleles of HLA-A33 and HLA-A 01.
Herein, a CTL "targets" a cell refers to the recognition by a CTL of a cell presenting a complex of HLA and a peptide of the present invention on its cell surface, and exhibits cytotoxic activity against the cell. Further, "specifically targeted" means that CTLs exhibit cytotoxic activity against these cells, but do not exhibit destructive activity against other cells. The expression "recognizing a cell" as used in the context of CTLs refers to binding via its TCR to a complex of HLA presented on the cell surface and a peptide of the present invention and exhibiting specific cytotoxic activity towards the cell. Thus, the CTLs of the invention are preferably CTLs capable of binding via the TCR to a complex formed between the peptide of the invention and HLA-A * 33:03) or HLA-A01 (more preferably HLA-A * 01:01) presented on the surface of a cell.
Furthermore, the CTL of the present invention is preferably a CTL induced by a method comprising the steps described in (a) or (b) below:
(a) Contacting in vitro CD8 positive T cells with APCs or exosomes presenting on their surface a complex of a peptide of the invention and HLA-A33 (more preferably HLA-A 33:03) or HLA-A01 (more preferably HLA-A 01:01), or
(B) A polynucleotide comprising subunits encoding each TCR capable of binding to a peptide of the invention presented on the cell surface by HLA-A33 (more preferably HLA-A 33:03) or HLA-A01 (more preferably HLA-A 01:01) is introduced into a CD8 positive T cell. The CTLs induced by this method have TCRs that specifically recognize complexes of peptides and HLA antigens for induction. Thus, they are cells having structural differences with other CTLs having different response specificities due to differences in TCR structure.
T Cell Receptor (TCR)
The invention also provides compositions comprising polynucleotides encoding each TCR subunit capable of binding to a peptide of the invention presented on the surface of a cell via HLA antigens, and methods of using the compositions. The polynucleotides confer specificity to CD8 positive T cells for FOXM1 expressing cancer cells by expressing TCRs capable of binding to the peptides of the invention presented on the surface of target cells of CD8 positive T cells by HLA antigens. Polynucleotides encoding the alpha and beta chains of the TCR subunits in CTLs induced by the peptides of the invention can be identified using methods known in the art (WO 2007/032555 and Morgan et al, J Immunol,171,3288 (2003)). For example, the PCR method is preferred for TCR analysis. Without being limited thereto, the PCR primer for analysis may be, for example, a primer set for amplification by combining the following 5 'side primer and 3' side primer:
5' side primer:
5' -R primer (5'-gtctaccaggcattcgcttcat-3') (SEQ ID NO: 62)
3' -Side primer:
TCR-alpha-chain C-region-specificity
3-TRa-C primer (5'-tcagctggaccacagccgcagcgt-3') (SEQ ID NO: 63)
TCR-beta-chain C1-region-specificity
3-TRb-C1 primer (5'-tcagaaatcctttctcttgac-3') (SEQ ID NO: 64) or
TCR-beta-chain C2-region-specificity
3-TR-beta-C2 primer (5'-ctagcctctggaatcctttctctt-3') (SEQ ID NO: 65)
TCRs formed by introducing the identified polynucleotides into CD8 positive T cells are capable of binding with high avidity to target cells presenting the peptides of the invention and mediating efficient killing of target cells presenting the peptides of the invention in vivo and in vitro.
The polynucleotides encoding each TCR subunit can be incorporated into a suitable vector, such as a retroviral vector. Such vectors are well known in the art. The polynucleotides or vectors comprising them may be introduced into CD8 positive T cells, e.g. CD8 positive T cells derived from a patient. The present invention provides a ready-to-use composition that allows for rapid modification of a patient's own T cells (or T cells derived from other subjects) to quickly and easily generate modified T cells with excellent cancer cell killing characteristics.
Herein, a specific TCR is a TCR that, when presented on the surface of a CD 8-positive T cell, is capable of conferring specific cytotoxic activity against the target cell by specifically recognizing a complex of the peptide of the invention and HLA antigen presented on the surface of the target cell. Specific recognition of the above complex can be confirmed by any known method, and preferable examples thereof include HLA multimeric staining analysis using HLA molecules and the peptide of the present invention, and ELISPOT assay method. By performing the ELISPOT assay, specific TCR-mediated recognition by T cells into which the above polynucleotide is introduced and target cells for intracellular signal transduction can be confirmed. When the above TCR is presented on the surface of a CD 8-positive T cell, it can also be confirmed by known methods whether the TCR is capable of conferring target cell-specific cytotoxic activity against a CD 8-positive T cell. Preferred methods include assaying for cytotoxic activity against HLA-positive target cells, for example, by chromium release assay or the like.
In the context of HLA-A33, the invention provides CTLs prepared by transforming CD8 positive T cells with polynucleotides encoding each TCR subunit (which bind, for example, to a complex formed by a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 and the HLA-A33 antigen).
In the context of HLA-A01, the present invention provides CTLs prepared by transforming CD8 positive T cells with polynucleotides encoding each TCR subunit (which bind, for example, to a complex formed by a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61 and an antigen of HLA-A 01).
The transformed CTLs are capable of homing (doming) in vivo (translocation from blood to lymphoid tissue (translocation)) and can be amplified using well-known in vitro culture methods (e.g., KAWAKAMI ET al, J immunol, 1989, 142:3252-61). The CTLs of the present invention can be utilized to form immunogenic compositions useful for the treatment or prevention (prevention) of disease in a patient in need of treatment and/or prevention (prevention) (for reference WO2006/031221, the contents of which are incorporated herein by reference).
IX. pharmaceutical compositions
The present invention also provides a composition or pharmaceutical composition comprising at least one active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC of the present invention;
(d) Exosomes of the invention, and
(E) The CTL of the present invention.
In addition to the above-described active ingredients, the pharmaceutical composition of the present invention may contain carriers, excipients, etc. as necessary, which are generally used for pharmaceuticals, without particular limitation. Examples of carriers that can be used in the pharmaceutical composition of the present invention include sterile water, physiological saline, phosphate buffer, culture solution, and the like. Accordingly, the present invention also provides a pharmaceutical composition comprising at least one active ingredient selected from the following (a) to (e) and a pharmaceutically acceptable carrier:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC of the present invention;
(d) Exosomes of the invention, and
(E) The CTL of the present invention.
In addition, the pharmaceutical composition of the present invention may contain stabilizers, suspensions, preservatives, surfactants, solubilizers, pH adjusters, aggregation inhibitors, and the like as needed.
FOXM1 expression was significantly up-regulated in cancer cells compared to normal tissues. The peptides of the invention or polynucleotides encoding the peptides may therefore be used to treat and/or prevent cancer, and/or prevent postoperative recurrence thereof. The present invention thus provides a pharmaceutical composition for the treatment and/or prevention of cancer, and/or for the prevention of postoperative recurrence thereof, comprising as an active ingredient one or more types of the peptide or polynucleotide of the present invention. Alternatively, the peptides of the invention may be prepared for presentation on the surface of exosomes or APCs for use as pharmaceutical compositions. Furthermore, CTLs of the present invention targeting any of the peptides of the present invention can also be used as the active ingredient of the pharmaceutical composition of the present invention. The pharmaceutical compositions of the present invention may comprise a therapeutically or pharmaceutically effective amount of the above-described active ingredients.
The pharmaceutical composition of the invention may also be used as a vaccine. In the context of the present invention, the term "vaccine" (also referred to as "immunogenic composition") refers to a composition that has the function of inducing an immune response that results in an anti-tumor effect when vaccinated into an animal. The pharmaceutical compositions of the invention are therefore useful for inducing an immune response that results in an anti-tumor effect. The immune response induced by the peptide, polynucleotide, APC, CTL and pharmaceutical composition of the present invention is not particularly limited as long as it is an immune response that causes an antitumor effect, and examples include induction of cancer cell-specific CTLs and induction of cancer cell-specific cytotoxic activities.
The pharmaceutical compositions of the invention are useful for treating and/or preventing cancer in a human subject or patient, and/or preventing postoperative recurrence thereof. The pharmaceutical composition of the present invention may be preferably used for subjects positive for at least one HLA selected from the group consisting of HLA-A33 and HLA-A 01. Furthermore, the pharmaceutical composition of the present invention can be preferably used for the treatment and/or prevention of cancers expressing FOXM1 and at least one HLa selected from HLA-A33 and HLA-A01 and/or the prevention of postoperative recurrence thereof.
In another embodiment, the present invention provides the use of an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
Or the present invention further provides an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
Or the present invention also provides a method or process for preparing a pharmaceutical composition for treating or preventing cancer, wherein the method or process comprises the step of formulating at least one active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
In another embodiment, the present invention also provides a method or process for preparing a pharmaceutical composition for treating or preventing cancer, wherein the method or process comprises the step of mixing an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
In another embodiment, the invention further provides a method for treating or preventing cancer comprising the step of administering to a subject at least one active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
In the present invention, peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs 1,2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 are identified as HLA-A33 restriction epitope peptides that induce potent and specific immune responses. Thus, pharmaceutical compositions of the invention comprising at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 are particularly suitable for administration to subjects having HLA-A33 (e.g. HLA-A 33: 03) as HLa antigen. The same applies to pharmaceutical compositions comprising polynucleotides encoding any of these peptides (i.e., polynucleotides of the invention), APCs or exosomes presenting these peptides (i.e., APCs or exosomes of the invention), or CTLs targeting these peptides (i.e., CTLs of the invention). That is, pharmaceutical compositions comprising an active ingredient associated with a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 are suitable for administration to a subject having HLA-A33 (i.e., a HLA-A33 positive subject). In a more preferred embodiment, the pharmaceutical composition of the invention is a pharmaceutical composition comprising a peptide having the amino acid sequence of SEQ ID NO. 2.
Similarly, in the present invention, peptides having amino acid sequences selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61 were identified as HLA-A01 restriction epitope peptides that can induce potent and specific immune responses. Thus, pharmaceutical compositions of the invention comprising at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61 are particularly suitable for administration to subjects having HLA-A01 (e.g. HLA-A 01: 01) as HLa antigen. The same applies to pharmaceutical compositions comprising polynucleotides encoding any of these peptides (i.e., polynucleotides of the invention). APC or exosomes presenting these peptides (i.e., APC or exosomes of the invention), or CTLs targeting these peptides (i.e., CTLs of the invention). That is, pharmaceutical compositions comprising an active ingredient associated with a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61 are suitable for administration to subjects having HLA-A01 (i.e., HLA-A01 positive subjects). In a more preferred embodiment, the pharmaceutical composition of the invention is a pharmaceutical composition comprising a peptide having the amino acid sequence of SEQ ID NO: 56.
The cancers treated and/or prevented by the pharmaceutical composition of the present invention are not particularly limited as long as they are FOXM 1-expressing cancers and include various cancers such as Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, testicular tumor, and the like. Furthermore, the pharmaceutical composition of the present invention may be preferably used for a subject homozygous or heterozygous for an HLA allele selected from HLA-A33 and HLA-A 01.
In addition to the above-described active ingredients, the pharmaceutical composition of the present invention may comprise other peptides having the ability to induce CTLs against cancer cells (e.g., other TAA-derived CTL inducing peptides), other polynucleotides encoding other peptides, other cells presenting other peptides, and the like.
The pharmaceutical compositions of the present invention may optionally further comprise other therapeutic substances as active ingredients, as long as they do not inhibit the antitumor effect of the above-mentioned active ingredients (e.g., the peptides of the present invention). For example, the pharmaceutical compositions of the present invention may optionally comprise anti-inflammatory compositions, analgesics, chemotherapeutic agents, and the like. In addition to including other therapeutic substances in the pharmaceutical compositions of the present invention themselves, the pharmaceutical compositions of the present invention may be administered sequentially or simultaneously with one or more other pharmaceutical compositions. The dosage of the pharmaceutical compositions of the invention and other pharmaceutical compositions depends on, for example, the type of pharmaceutical composition used and the disease to be treated, as well as the timing (schedule) and route of administration.
It will be appreciated that the pharmaceutical compositions of the present invention may comprise other components conventional in the art, in addition to the ingredients explicitly mentioned herein, in view of the type of formulation.
The invention also provides an article of manufacture or kit comprising the pharmaceutical composition of the invention. The article of manufacture or kit of the invention may comprise a container containing the pharmaceutical composition of the invention. Examples of suitable containers include, but are not limited to, bottles, vials or test tubes. The container may be made of a variety of materials, such as glass or plastic. The label may be attached to the container and the disease or disease state to which the pharmaceutical composition of the invention is applied may be described in the label. The label may also indicate instructions regarding administration, and so forth.
In addition to the container containing the pharmaceutical composition of the invention, preferably the article or kit of the invention may optionally further comprise a second container containing a pharmaceutically acceptable diluent. The articles or kits of the invention may also include other materials required from a commercial standpoint and from a user's perspective, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
If desired, the pharmaceutical compositions of the present invention may be provided packaged in a pharmaceutical pack or dispenser device which may be filled with one or more unit dosage forms containing the active ingredient. For example, the pack may comprise, for example, a metal or plastic foil, such as a blister pack. The pack or dispensing device may be accompanied by instructions for administration.
(1) Pharmaceutical composition comprising peptide as active ingredient
Pharmaceutical compositions comprising the peptides of the invention may be formulated as desired by conventional formulation methods. In addition to the peptide of the present invention, the pharmaceutical composition of the present invention may contain carriers, excipients, etc. commonly used in medicines, without particular limitation. Examples of carriers that can be used in the pharmaceutical composition of the present invention include sterilized water (e.g., water for injection), physiological saline, phosphate buffer, phosphate buffered saline, tris buffered saline, 0.3% glycine, culture solution, and the like. In addition, the pharmaceutical composition of the present invention may contain stabilizers, suspending agents, preservatives, surfactants, solubilizers, pH adjusting agents, aggregation inhibitors, and the like as needed. The pharmaceutical composition of the present invention can induce specific immunity against FOXM 1-expressing cancer cells, and thus can be used for the purpose of cancer treatment or prevention (prevention).
For example, the pharmaceutical composition of the present invention can be prepared by dissolving in a pharmaceutically or physiologically acceptable water-soluble carrier such as sterilized water (e.g., water for injection), physiological saline, phosphate buffer, phosphate buffered saline, and Tris buffered saline, and adding a stabilizer, a suspending agent, a preservative, a surfactant, a solubilizing agent, a pH adjustor, an aggregation inhibitor or the like as needed, and then sterilizing the peptide solution. The method for sterilizing the peptide solution is not particularly limited, and is preferably performed by filtration sterilization. The filter sterilization may be performed using, for example, a filter sterilization filter having a pore size of 0.22 μm or less. The filter sterilized peptide solution may be administered to a subject, for example, as an injection, but is not limited thereto.
The pharmaceutical composition of the present invention can be prepared as a lyophilized formulation by lyophilizing the above peptide solution. The lyophilized preparation can be prepared by filling the peptide solution prepared as described above into a suitable container such as an ampoule, vial or plastic container, followed by lyophilization and encapsulation into the container after pressure recovery with a washed sterilized rubber stopper or the like. The lyophilized formulation may be administered to a subject after it is redissolved in a pharmaceutically or physiologically acceptable water-soluble carrier such as sterilized water (e.g., water for injection), physiological saline, phosphate buffer, phosphate buffered saline, tris buffered saline, and the like. Preferred examples of the pharmaceutical composition of the present invention include an injectate of such a filter-sterilized peptide solution and a lyophilized formulation resulting from lyophilizing the peptide solution. The invention also includes kits containing such freeze-dried formulations and re-dissolution solutions. The invention also includes a container containing a lyophilized formulation (which is a pharmaceutical composition of the invention) and a kit containing a container of a re-dissolving solution.
The pharmaceutical composition of the present invention may comprise a combination of two or more types of the peptides of the present invention. The combination of peptides may take the form of a cocktail of mixed peptides, or may be conjugated to each other using standard techniques. For example, peptides may be chemically linked or expressed as a single fusion polypeptide. By administering the peptide of the present invention, the peptide is displayed on the APC by the HLA antigen at a high density, and then CTL that specifically reacts with a complex formed between the displayed peptide and the HLA antigen is induced. Alternatively, APCs (e.g., DCs) can be isolated from a subject and then stimulated with the peptides of the invention to obtain APCs displaying any of the peptides of the invention on their cell surfaces. Re-administering these APCs to a subject to induce CTLs in the subject, as a result, aggressiveness against FOXM 1-expressing cancer cells can be improved.
The pharmaceutical compositions of the present invention may also contain adjuvants known to be effective in the establishment of cellular immunity. An adjuvant refers to a compound that enhances an immune response against an antigen that is immunologically active when administered together (or sequentially) with the antigen. Known adjuvants described in the literature, for example Clin Microbiol Rev 1994,7:277-89, can be used. Examples of suitable adjuvants include, but are not limited to, aluminum salts (aluminum phosphate, aluminum hydroxide, aluminum oxyhydroxide, etc.), alum, cholera toxin, salmonella toxin, IFA (incomplete freund's adjuvant), CFA (complete freund's adjuvant), ISCOMatrix, GM-CSF and other immunostimulatory cytokines, oligodeoxynucleotides containing CpG motifs (CpG 7909, etc.), oil-in-water emulsions, saponins or derivatives thereof (QS 21, etc.), lipopolysaccharides such AS lipid a or derivatives thereof (MPL, RC529, GLA, E6020, etc.), lipopeptides, lactoferrin, flagellin, double stranded RNA or derivatives thereof (poliic, etc.), bacterial DNA, imidazoquinolines (Imiquimod, R848, etc.), C-lectin ligands (trehalose-6, 6' -bisbehenic acid/ester (TDB, etc.), CD1d ligands (alpha-galactoceramide, etc.), squalene emulsions (MF 59, AS03, AF03, etc.), PLGA, etc. In a kit comprising the pharmaceutical composition of the invention, the adjuvant may be contained in another container separate from the pharmaceutical composition comprising the peptide of the invention. In this case, the adjuvant and the pharmaceutical composition may be administered to the subject continuously or mixed together immediately prior to administration to the subject. The invention also provides such kits comprising a peptide pharmaceutical composition comprising the invention and an adjuvant. When the pharmaceutical composition of the present invention is a lyophilized formulation, the kit may further comprise a redissolution solution. Furthermore, the present invention provides a kit comprising a container containing the pharmaceutical composition of the present invention and a container storing an adjuvant. The kit may further comprise a container for storing the redissolved solution, as desired.
When an oil adjuvant is used as an adjuvant, the pharmaceutical composition of the present invention may be prepared as an emulsion. For example, an emulsion may be prepared by mixing and stirring the peptide solution prepared as described above and an oil adjuvant. The peptide solution may be a solution that resolubilizes after lyophilization. The emulsion may be a W/O type emulsion and an O/W type emulsion, and the W/O type emulsion is preferably used for obtaining a high immune response enhancing effect. IFA may be preferably used as an oil adjuvant, but is not limited thereto. The preparation of the emulsion may be carried out immediately prior to administration to a subject, and in such a case, the pharmaceutical composition of the present invention may be provided as a kit comprising the peptide solution of the present invention and an oily adjuvant. When the pharmaceutical composition of the present invention is a lyophilized formulation, the kit may further comprise a redissolution solution.
Furthermore, the pharmaceutical composition of the present invention may be a liposome formulation in which the peptide of the present invention is encapsulated, a particle formulation in which the peptide is bound to beads having a diameter of several micrometers, or a formulation in which the lipid is bound to the peptide.
In another embodiment of the invention, the peptides of the invention may also be administered in the form of pharmaceutically acceptable salts. Examples of preferred salts include salts with alkali metals (lithium, potassium, sodium, etc.), salts with alkaline earth metals (calcium, magnesium, etc.), salts with other metals (copper, iron, zinc, manganese, etc.), salts with organic bases, salts with amines, salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, etc.), and salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, nitric acid, etc.). The phrase "pharmaceutically acceptable salt" as used herein refers to salts that retain the biological, physiological, pharmacological and pharmaceutical effectiveness and properties of the compound. Accordingly, pharmaceutical compositions comprising pharmaceutically acceptable salts of the peptides of the invention are also included in the invention. Furthermore, "the peptide of the present invention" includes pharmaceutically acceptable salts thereof in addition to the free peptide.
In some embodiments, the pharmaceutical compositions of the present invention may further comprise a component that elicits (prime) CTLs. Lipids have been identified as components capable of eliciting CTLs against viral antigens in vivo. For example, palmitic acid residues may be linked to epsilon-and alpha-amino groups of lysine residues and then to peptides of the invention. The lipidated peptide may then be administered directly in micelles or particles, incorporated into liposomes, or emulsified in an adjuvant. As other examples of lipid-initiated CTL responses, E.coli lipoproteins, such as tripalmitoyl-S-glyceryl cysteinyl serinyl-serine (P3 CSS), can be used to trigger CTL when covalently linked to a suitable peptide (see, e.g., deres et al Nature 1989, 342:561-4).
Examples of suitable methods for administering the peptides or pharmaceutical compositions of the invention include, but are not limited to, oral, intradermal, subcutaneous, intramuscular, intraosseous, intraperitoneal, and intravenous injection, as well as systemic or topical administration to the vicinity of a target site. The preferred method of administration includes subcutaneous injection into the vicinity of a lymph node such as the axilla or groin. More preferably, for example, when the pharmaceutical composition of the present invention comprises a peptide or exosomes as an active ingredient, subcutaneous administration is preferred. Alternatively, the composition having APC or CTL as an active ingredient may be administered by intravenous injection or the like. Administration may be by single administration or by boosting by multiple administrations.
The peptides of the invention can be administered to a subject in a therapeutically or pharmaceutically effective amount to treat cancer, or in a therapeutically or pharmaceutically effective amount to induce immunity (more specifically CTLs) against FOXM 1-expressing cancer cells. The dosage of the peptide of the present invention may be appropriately adjusted depending on the disease to be treated, the age, weight, method of administration, etc. of the patient. For each peptide of the invention, the dosage is typically 0.001mg to 1000mg, for example 0.01mg to 100mg, for example 0.1mg to 30mg, for example 0.1mg to 10mg, for example 0.5mg to 5mg. The dosing interval may be once every few days to a few months, and for example, dosing may be performed at weekly intervals. The person skilled in the art can suitably select the appropriate dose (dose) (dosage).
In a preferred embodiment, the pharmaceutical composition of the invention comprises a therapeutically effective amount of a peptide of the invention and a pharmaceutically or physiologically acceptable carrier. In another embodiment, the pharmaceutical composition of the invention comprises a therapeutically effective amount of a peptide of the invention, a pharmaceutically or physiologically acceptable carrier and an adjuvant. The pharmaceutical composition of the invention may comprise from 0.001mg to 1000mg, preferably from 0.01mg to 100mg, more preferably from 0.1mg to 30mg, even more preferably from 0.1mg to 10mg, for example from 0.5mg to 5mg of the peptide of the invention. When the pharmaceutical composition of the present invention is an injection, it may contain the peptide of the present invention at a concentration of 0.001mg/ml to 1000mg/ml, preferably 0.01mg/ml to 100mg/ml, more preferably 0.1mg/ml to 30mg/ml, even more preferably 0.1mg/ml to 10mg/ml, for example 0.5mg/ml to 5mg/ml. In this case, for example, 0.1 to 5ml, preferably 0.5ml to 2ml of the pharmaceutical composition of the present invention may be administered to a subject by injection.
Furthermore, the present invention provides a method of treating and/or preventing cancer and/or preventing postoperative recurrence thereof, comprising administering to a subject a therapeutically effective amount of a peptide of the present invention or a pharmaceutical composition of the present invention. As described above, the peptides of the invention may be administered to a subject in a single dose, typically in an amount of 0.001mg to 1000mg, e.g., 0.01mg to 100mg, e.g., 0.1mg to 30mg, e.g., 0.1mg to 10mg, or e.g., 0.5mg to 5mg. In a preferred embodiment, the peptide of the invention is administered to a subject with an adjuvant. Furthermore, the dosing interval may be once every few days to a few months, preferably once every few days to a month, for example once a week or once every two weeks.
(2) Pharmaceutical composition containing polynucleotide as active ingredient
The pharmaceutical compositions of the invention may also contain polynucleotides encoding the peptides disclosed herein in an expressible form. In this context, the phrase "in an expressible form" means that the polynucleotide, when introduced into a cell, will express the peptide of the invention. In an exemplary embodiment, the sequence of the polynucleotide of the invention includes regulatory elements necessary for expression of the peptide of the invention. The polynucleotides of the invention may have sequences required to achieve stable insertion into the genome of the target Cell (see, e.g., thomas KR & CAPECCHI MR, cell 1987,51:503-12 for a description of homologous recombination cassette vectors). See, e.g., wolff et al, science 1990,247:1465-8, U.S. Pat. No. 5,580,859,5,589,466,5,804,566,5,739,118,5,736,524,5,679,647, and WO98/04720. Examples of DNA-based delivery techniques include "naked DNA", facilitated (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).
The peptides of the invention may also be expressed using viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccinia or avipox. For example, vaccinia virus can be used as a vector for expressing the peptide of the present invention. After introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide and thereby elicits an immune response. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Pat. No. 4,722,848. Another carrier is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al, nature 1991,351:456-60. Various other vectors are apparent which can be used for therapeutic administration or immunization, such as adenovirus and adeno-associated virus vectors, retrovirus vectors, salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., shata et al, mol Med Today 2000,6:66-71; sheldlock et al, J Leukoc Biol2000,68:793-806; hipp et al, in Vivo 2000,14:571-85.
Delivery of the polynucleotide of the invention to a patient may be direct, in which case the patient may be directly exposed to the vector carrying the polynucleotide of the invention, or indirect, in which case the cells are first transformed in vitro with the vector carrying the polynucleotide of the invention and then transplanted into the patient. These two approaches are called in vivo and ex vivo gene therapy, respectively.
For a general review of methods of gene therapy see Goldspiel et al, CLINICAL PHARMACY1993,12:488-505; wu and Wu,Biotherapy 1991,3:87-95;Tolstoshev,Ann Rev Pharmacol Toxicol 1993,33:573-96;Mulligan,Science 1993,260:926-32;Morgan&Anderson,Ann Rev Biochem 1993,62:191-217;Trends in Biotechnology 1993,11(5):155-215. describe well known methods in the art of recombinant DNA technology that can also be applied to the present invention in Ausubel et al, current Protocols in Molecular Biology, john Wiley & Sons, NY,1993; and Krieger, GENE TRANSFER AND Expression, A Laboratory Manual, stockton Press, NY, 1990.
Similar to administration of the peptide, administration of the polynucleotide may be by oral, intradermal, subcutaneous, intravenous, intramuscular, intraosseous, or intraperitoneal injection, or the like. The administration of the polynucleotide may be systemic or local to the vicinity of the target site. Administration may be performed by a single administration or may be enhanced by multiple administrations. The polynucleotides of the invention may be administered to a subject in a therapeutically or pharmaceutically effective amount for inducing immunity (more specifically, CTLs) against FOXM 1-expressing cancer cells, or in a therapeutically or pharmaceutically effective amount for treating cancer. The dosage of the polynucleotide in a suitable vector or the dosage of the polynucleotide in a cell transformed with the polynucleotide encoding the peptide of the present invention may be appropriately adjusted depending on the disease to be treated, the age, weight of the patient, the method of administration, and the like, and is generally 0.001mg to 1000mg, for example, 0.01mg to 100mg, for example, 0.1mg to 30mg, for example, 0.1mg to 10mg, or for example, 0.5mg to 5mg. The dosing interval may be once every few days to a few months, for example, dosing may be at weekly intervals. The person skilled in the art can suitably select the appropriate dose (dose) (dosage).
Methods of using peptides, exosomes, APCs and CTLs
The peptides and polynucleotides of the invention are useful for inducing APCs and CTLs. Exosomes and APCs of the invention may also be used to induce CTLs. Peptides, polynucleotides, exosomes and APCs may be used in combination with any other compound as long as their CTL inducibility is not inhibited. Thus, the CTL of the invention can be induced using a pharmaceutical composition comprising any of the peptides, polynucleotides, APCs and exosomes of the invention. In addition, the APCs of the present invention can be induced using a pharmaceutical composition comprising the peptides or polynucleotides of the present invention.
(1) Method for inducing APC
The present invention provides a method for inducing APCs having CTL inducibility using the peptide or polynucleotide of the present invention.
The method of the invention comprises the step of contacting the APC with the peptide of the invention in vitro, ex vivo or in vivo. For example, a method of contacting an APC with a peptide ex vivo may comprise the steps of:
(a) Collecting APC from the subject, and
(B) Contacting the APC of step (a) with a peptide of the present invention.
The above APC is not limited to a specific kind of cells, and cells known to present protein antigens on the cell surface thereof to be recognized by lymphocytes, such as DC, langerhans cells, macrophages, B cells, and activated T cells, may be used. DCs have the strongest CTL inducibility among APCs, and thus it is preferable to use DCs. Any peptide of the invention may be used by itself or in combination with other peptides of the invention. Furthermore, the peptides of the invention may be used in combination with other CTL-inducing peptides (e.g., other TAA-derived CTL-inducing peptides).
Meanwhile, when the peptide of the present invention is administered to a subject, APCs are contacted with the peptide in vivo, and as a result, APCs having high CTL inducibility are induced in the body of the subject. Thus, the methods of the invention may comprise the step of administering the peptide of the invention to a subject. Similarly, when the polynucleotide of the present invention in an expressible form is administered to a subject, the peptide of the present invention is expressed in vivo, and the expressed peptide is contacted with APCs in vivo, as a result, APCs having high CTL inducibility are induced in the body of the subject. Thus, the invention may further comprise the step of administering a polynucleotide of the invention to a subject.
In order to induce an APC having CTL-inducing ability, the present invention further includes a step of introducing the polynucleotide of the present invention into the APC. For example, the method may comprise the steps of:
(a) Collecting APC from the subject, and
(B) Introducing a polynucleotide encoding a peptide of the invention into the APC of step (a).
Step (b) may be performed as described in section "vi.
Accordingly, in one embodiment, the present invention provides a method of inducing APC having CTL inducibility, comprising the following steps (a) or (b):
(a) Contacting an APC with a peptide of the invention, and
(B) A polynucleotide encoding a peptide of the present invention is introduced into an APC.
Furthermore, the present invention provides a method for producing an APC having CTL inducibility, which comprises the following step (a) or (b):
(a) Contacting an APC with a peptide of the invention, or
(B) A polynucleotide encoding a peptide of the present invention is introduced into an APC.
The above methods may be carried out in vitro, ex vivo or in vivo, and preferably they are carried out in vitro or ex vivo. The APCs used in the above methods may be derived from a subject to whom the induced APCs are intended to be administered, or they may be derived from a different subject. When APCs from a subject (donor) different from the subject to be administered are used, the subject to be administered and the donor must have the same HLA type.
In the method of the present invention, when a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 or a modified peptide thereof is used as the peptide of the present invention, HLA in both the subject and the donor is preferably HLA-a33 (more preferably HLA-a 33:03). Or the APC used in the above method is preferably an APC expressing HLA-A33 (more preferably HLA-A 33:03).
Similarly, when a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61 or a modified peptide thereof is used as the peptide of the present invention, HLA in the subject and the donor is preferably HLA-a01 (more preferably HLA-a 01: 01). Or the APC used in the above method is preferably an APC expressing HLA-A01 (more preferably HLA-A 01: 01). After separating PBMCs from donor-collected blood by a specific gravity centrifugation method or the like, APCs may be prepared from PBMCs using known methods.
In another embodiment, the present invention also provides a pharmaceutical composition comprising the peptide of the present invention or a polynucleotide encoding the peptide for inducing APC having CTL inducibility.
Or the present invention also provides the use of the peptide of the present invention or a polynucleotide encoding the peptide for preparing a pharmaceutical composition for inducing APC having CTL inducibility.
Or the present invention also provides the peptide of the present invention or a polynucleotide encoding the same for inducing APC having CTL inducibility.
Or the invention further provides a method or process for preparing a pharmaceutical composition for inducing APC, wherein the method or process comprises the step of formulating the peptide of the invention or the polynucleotide of the invention with a pharmaceutically or physiologically acceptable carrier.
In another embodiment, the present invention further provides a method or process for preparing a pharmaceutical composition for inducing APCs having CTL inducibility, wherein the method or process comprises the step of mixing the peptide of the present invention or the polynucleotide of the present invention with a pharmaceutically or physiologically acceptable carrier.
APCs induced by the method of the present invention can induce CTLs specific for FOXM1 (i.e., CTLs of the present invention).
(2) Method for inducing CTL
The present invention also provides a method for inducing CTL using the peptide, polynucleotide, exosome or APC of the present invention. The invention also provides methods of inducing CTLs using one or more polynucleotides encoding polypeptides capable of forming T Cell Receptors (TCRs) (i.e., TCR subunits) capable of recognizing complexes of the peptides of the invention and HLA antigens. Preferably, the method of inducing CTLs comprises at least one step selected from the group consisting of:
(a) Contacting CD8 positive T cells with antigen presenting cells that present on their surface a complex of HLA antigens and a peptide of the present invention;
(b) Contacting a CD 8-positive T cell with exosomes presenting on their surface a complex of an HLA antigen and a peptide of the invention, and
(C) Introducing into a CD8 positive T cell one or more polynucleotides encoding polypeptides capable of forming a TCR capable of recognizing a complex of a peptide of the invention and an HLA antigen.
When the peptide, polynucleotide, APC or exosome of the present invention is administered to a subject, CTLs are induced in the body of the subject, and the intensity of an immune response targeting FOXM 1-expressing cancer cells is enhanced. Thus, the methods of the invention can include the step of administering the peptide, polynucleotide, APC, or exosome of the invention to a subject.
Alternatively, they may be used in vitro or ex vivo to induce CTLs. For example, the method of the present invention may comprise the steps of:
(a) Collecting APCs from a subject;
(b) Contacting the APC of step (a) with a peptide of the invention, and
(C) Co-culturing the APC of step (b) with CD8 positive T cells.
The induced CTLs can then be returned to the subject.
The APCs to be co-cultured with CD8 positive T cells in step (c) above can also be prepared by introducing a polynucleotide encoding a peptide of the invention into the APCs as described in the section "vi. However, the APCs used in the method of the present invention are not limited thereto, and any APCs presenting complexes of HLA antigens and the peptide of the present invention on the surface thereof may be used.
In the method of the present invention, in addition to such APCs, exosomes presenting on their surface a complex formed by HLA antigen and the peptide of the present invention can also be used. That is, the method of the present invention may include a step of co-culturing with exosomes presenting complexes of HLA antigens and the peptides of the present invention on their surfaces. Such exosomes may be prepared by the methods described in the "v. exosomes" section above.
Furthermore, CTLs can be induced by introducing a vector comprising a polynucleotide encoding each subunit of a TCR capable of binding to the peptide of the invention presented by HLA antigens on the cell surface into CD8 positive T cells. Such transformation may be performed as described in the section "viii.t cell receptor (TCR)", above.
Accordingly, in one embodiment, the present invention provides a method of inducing CTLs, comprising the steps selected from the group consisting of:
(a) Co-culturing CD8 positive T cells with APCs that present on their surface a complex of HLA antigens and the peptides of the invention;
(b) Co-culturing CD8 positive T cells with exosomes presenting complexes of HLA antigens and the peptides of the invention on their surface, and
(C) A vector comprising a polynucleotide encoding each subunit of a TCR capable of binding to a peptide of the invention presented by HLA antigens on the cell surface is introduced into a CD8 positive T cell.
The above methods may be carried out in vitro, ex vivo or in vivo, and preferably they are carried out in vitro or ex vivo. The APCs or exosomes and CD8 positive T cells used in the above methods may be derived from the subject to whom the induced CTLs are intended to be administered, or they may be derived from different subjects. When using APC or exosomes and CD8 positive T cells from a subject (donor) different from the subject to be administered, the subject and the donor must have the same HLA type. For example, when peptides having the amino acid sequences of SEQ ID NOs:1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 or modified peptides thereof are used as the peptides of the present invention, HLA type is preferably HLA-a33 (more preferably HLA-a 33:03) in the administration subject and donor. Or the APC or exosome used in the above method is preferably an APC or exosome presenting on its surface a complex of HLA-A33 (more preferably HLA-A 33:03) and a peptide of the invention (a peptide having the amino acid sequence of SEQ ID NOs:1, 2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 or a modified peptide thereof). In this case, the induced CTLs show cytotoxic activity against cells presenting a complex of HLA-A33 and the peptide of the present invention (e.g., HLA-A33 positive cells expressing FOXM 1).
Or, for example, when a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61 or a modified peptide thereof is used as the peptide of the present invention, HLA type in both the subject and the donor is preferably HLA-a01 (more preferably HLA-a 01: 01). Or the APC and exosomes used in the above method are preferably APC and exosomes presenting a complex of HLA-A01 (more preferably HLA-A x 01: 01) and a peptide of the invention (a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61 or a modified peptide thereof) on their surface. In this case, the induced CTLs show cytotoxic activity against cells presenting a complex of HLA-A01 and the peptide of the present invention (e.g., HLA-A01 positive cells expressing FOXM 1).
In another embodiment, the present invention also provides a composition or pharmaceutical composition for inducing CTLs, comprising at least one active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface, and
(D) Exosomes presenting the peptides of the invention on their surface.
In another embodiment, the present invention also provides the use of an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface, and
(D) Exosomes presenting the peptides of the invention on their surface.
Or the present invention further provides an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface, and
(D) Exosomes presenting the peptides of the invention on their surface.
Or the present invention further provides a method or process for preparing a composition or pharmaceutical composition for inducing CTLs, which is a method or process comprising the step of formulating an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface, and
(D) Exosomes presenting the peptides of the invention on their surface.
In another embodiment, the present invention further provides a method or process for preparing a composition or pharmaceutical composition for inducing CTLs, comprising the step of mixing an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface, and
(D) Exosomes presenting the peptides of the invention on their surface.
XI method for inducing immune response
The invention also provides methods of inducing an immune response against FOXM 1-expressing cancers. Suitable cancers include, but are not limited to, acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, testicular tumor, and the like. Preferably, the cancer expresses at least one HLA selected from the group consisting of HLA-A33 and HLA-A 01.
The invention also provides methods of inducing an immune response against FOXM 1-expressing cancer cells. FOXM1 is thought to be overexpressed in the various types of cancers described above. Thus, when an immune response against FOXM 1-expressing cancer cells is induced, proliferation of the cancer cells is inhibited as a result. Accordingly, the present invention also provides a method of inhibiting the proliferation of FOXM 1-expressing cancer cells. The methods of the invention are particularly useful for inhibiting proliferation of cancer cells expressing FOXM1 and at least one HLa selected from HLA-A33 and HLA-A 01.
The method of the invention may comprise the step of administering a composition comprising any of the peptides of the invention or polynucleotides encoding said peptides. The methods of the invention also encompass the administration of APCs or exosomes presenting any of the peptides of the invention. See section "IX. pharmaceutical compositions" for details, particularly for a description of the use of the pharmaceutical compositions of the invention as vaccines. In addition, exosomes and APCs useful in the methods of inducing an immune response of the present invention are described in detail in items (1) and (2) above "v. Exosomes", "vi. Antigen Presenting Cells (APCs)", and "x. Methods of using peptides, exosomes, APCs, and CTLs".
In another embodiment, the invention provides a pharmaceutical composition or vaccine for inducing an immune response against FOXM 1-expressing cancer, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
Or the invention also provides a pharmaceutical composition or vaccine for inducing an immune response against FOXM 1-expressing cancer cells, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
Or the invention also provides a pharmaceutical composition or vaccine for inhibiting the proliferation of FOXM 1-expressing cancer cells, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
In another embodiment, the invention provides the use of an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
Or the invention also provides the use of an active ingredient selected from the following in the manufacture of a pharmaceutical composition or vaccine for inducing an immune response against FOXM1 expressing cancer cells:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
Or the invention further provides the use of an active ingredient selected from the following for the preparation of a pharmaceutical composition or vaccine for inhibiting proliferation of FOXM 1-expressing cancer cells:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
The invention also provides a method or process for preparing a pharmaceutical composition for inducing an immune response against FOXM 1-expressing cancer, wherein the method may comprise the step of mixing or formulating the peptide or polynucleotide of the invention with a pharmaceutically acceptable carrier.
Or the present invention provides a method of inhibiting proliferation of FOXM 1-expressing cancer cells or a method of inducing an immune response against cancer, comprising the step of administering to a subject a vaccine or pharmaceutical composition comprising an active ingredient selected from the group consisting of:
(a) The peptides of the invention;
(b) Polynucleotides encoding the peptides of the invention in an expressible form;
(c) APC presenting the peptide of the present invention on its surface;
(d) Exosomes presenting the peptides of the invention on their surface, and
(E) The CTL of the present invention.
In the context of the present invention, FOXM1 expressing cancers may be treated by administering the peptides, polynucleotides, APCs, exosomes and/or CTLs of the present invention. Or by administering the peptides, polynucleotides, APCs, exosomes and/or CTLs of the present invention can induce an immune response against FOXM1 expressing cancers. Examples of such cancers include Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, testicular tumor, and the like, but are not limited thereto. Furthermore, immune responses against FOXM 1-expressing cancers can be induced by administration of the peptides, polynucleotides, APCs, exosomes and/or CTLs of the present invention. Thus, prior to administration of a vaccine or pharmaceutical composition comprising the above-described active ingredient, it is preferable to confirm whether the FOXM1 expression level at the diseased site in the subject to be treated is increased.
Accordingly, in one embodiment, the present invention provides a method of treating FOXM 1-expressing cancer in a patient in need of cancer treatment, wherein the method comprises the steps of:
(i) Measuring the level of FOXM1 expression in a biological sample collected from a diseased site of a subject having cancer;
(ii) Identifying a subject having a FOXM 1-expressing cancer based on the FOXM1 expression level measured in (i), and
(Iii) Administering at least one component selected from the group consisting of (a) to (e) above to a patient suffering from a cancer that overexpresses FOXM1 compared to a normal control.
Or the present invention further provides vaccines and pharmaceutical compositions comprising at least one active ingredient selected from the group consisting of (a) to (e) above for administration to a patient having cancer expressing FOXM 1. The present invention also provides a method of identifying or selecting a subject to be treated with at least one active ingredient selected from the group consisting of (a) to (e) above, wherein the method comprises the steps of:
(i) Measuring the level of FOXM1 expression in a biological sample collected from a diseased site of a subject having cancer;
(ii) Identifying a subject having a FOXM 1-expressing cancer based on the FOXM1 expression level measured in (i), and
(Iii) Identifying or selecting the subject identified in (ii) as a subject that can be treated with at least one active ingredient selected from the group consisting of (a) to (e).
The biological sample for measuring FOXM1 expression level collected from the subject in the above-described method is not particularly limited, and for example, a tissue sample containing cancer cells collected by biopsy or the like can be preferably used. The expression level of FOXM1 in a biological sample can be measured by a known method, for example, a method of detecting a transcript of FOXM1 gene by a probe or PCR method (e.g., cDNA microarray method, northern blotting method, RT-PCR method, etc.), a method of detecting a translational product of FOXM1 gene by an antibody, etc. (e.g., western blotting method, immunostaining method, etc.), and the like can be used. Further, the biological sample may be a blood sample, and in this case, the blood level of an antibody against FOXM1 or a fragment thereof is measured, and the FOXM1 expression level of the affected part may be estimated based on the blood level. The blood level of an antibody against FOXM1 may be measured using a known method, for example, an Enzyme Immunoassay (EIA), an enzyme-linked immunosorbent assay (ELISA), a Radioimmunoassay (RIA), or the like using FOXM1 protein or the peptide of the present invention as an antigen may be used.
In general, in tissues and cells that do not express FOXM1, few FOXM1 transcripts and translation products were detected. Thus, when the transcript or the translation product of FOXM1 is detected in a cancer cell or a tissue sample containing a cancer cell collected from a subject, cancer expression of FOXM1 in the subject can be determined. In blood samples of subjects without FOXM1 expressing cancer, little antibodies to FOXM1 or fragments thereof were detected. Thus, when antibodies to FOXM1 or fragments thereof are detected in a blood sample collected from a subject, cancer expression of FOXM1 in the subject can be determined.
Whether or not FOXM1 is expressed by a subject's cancer may also be compared to measurements of the same type of biological material collected from a non-cancer site of the subject or to the same type of biological material collected from a subject not having cancer (normal control sample). That is, when the level in the biological sample of the test subject is elevated in comparison to the measured target level in the normal control sample (normal control level), the cancer of the subject is assessed as expressing FOXM1. For example, when the amount of the detected measurement object is increased by at least 10% or more compared to a normal control level, the cancer expression FOXM1 of the subject can be evaluated. Desirably, the amount of the detected measurement target is preferably 25% or more, more preferably 50% or more, higher than the normal control level. In addition, the amount of the detected transcript or translation product of FOXM1 can be assessed by normalization to the amount of a known housekeeping gene (e.g., β -actin, glyceraldehyde-3-phosphate dehydrogenase, or ribosomal protein P1) detected.
In a preferred embodiment, the HLA type of the subject is preferably confirmed prior to administration of at least one active ingredient selected from the group consisting of (a) to (e) above. For example, for subjects to be administered an active ingredient associated with a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 6,7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46, HLA-A33 positive subjects are preferably selected. For subjects to be administered an active ingredient related to a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61, preference is given to HLA-A01 positive subjects.
The invention also provides complexes of the peptides of the invention with HLA. The complex of the present invention described above may be a monomer or a polymer. When the composite of the present invention is a polymer, the number of polymerization is not particularly limited, and may be any number of polymerization polymers. Examples include, but are not limited to, tetramers, pentamers, hexamers, and the like. The multimers of the invention also include dextramers (WO 2002/072631) and streptamers (Knabel M et al, nat Med.2002Jun;8 (6): 631-7.). Complexes of peptides of the invention and HLA can be prepared according to known methods (e.g., altman JD et al, science 1996,274 (5284): 94-6; WO2002/072631; WO2009/003492;Knabel M,Nat Med.2002Jun;8 (6): 631-7, etc.).
For example, the complexes of the invention can be used to quantify CTLs specific for the peptides of the invention. For example, a blood sample is collected from a subject administered the pharmaceutical composition of the invention, and after isolation of PBMCs, CD4 negative cells are prepared and contacted with the fluorescent dye-conjugated complex of the invention. The percentage of CTLs specific for the peptides of the invention can then be measured by flow cytometry analysis. For example, the immune response induction of the pharmaceutical composition of the present invention can be monitored by measuring specific CTLs against the peptide of the present invention before, during and/or after administration of the pharmaceutical composition of the present invention.
XII antibodies
The invention further provides antibodies that bind to the peptides of the invention. Preferred antibodies specifically bind to the peptides of the invention and do not bind (or bind weakly) to the peptides of the invention. In another embodiment, such antibodies may include antibodies that recognize peptides in the context of HLA molecules, i.e., antibodies that bind to peptide-MHC complexes. The binding specificity of an antibody can be confirmed by an inhibition assay. That is, if the binding between the antibody to be analyzed and the full-length FOXM1 polypeptide is inhibited in the presence of the peptide of the present invention, the antibody shows specific binding to the peptide of the present invention. Antibodies to the peptides of the invention are useful in disease diagnosis and prognosis assays, as well as in selecting subjects for administration of the pharmaceutical compositions of the invention for selection and monitoring of the pharmaceutical compositions of the invention.
The invention also provides various immunoassays for detecting and/or quantifying the peptides of the invention or fragments thereof. Such immunoassays include, but are not limited to, radioimmunoassays, immunochromatography, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescence assays (ELIFA), and the like, and are performed within the scope of various immunoassay formats well known in the art.
The antibodies of the invention can be used in immunological imaging methods that can detect FOXM 1-expressing cancers, and examples include, but are not limited to, radioscintillation imaging using the labeled antibodies of the invention. Such assays are used clinically to detect, monitor and prognose cancers expressing FOXM1, and examples of such cancers include, but are not limited to, acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, testicular tumor, and the like.
The antibody of the present invention may be used in any arbitrary form, for example, a monoclonal or polyclonal antibody, and may further include antisera obtained by immunizing an animal (such as a rabbit) with the peptide of the present invention, all kinds of polyclonal and monoclonal antibodies, human antibodies, and chimeric and humanized antibodies produced by genetic recombination.
The peptide of the present invention or a fragment thereof used as an antigen to obtain an antibody can be obtained by chemical synthesis or genetic engineering techniques based on the amino acid sequences disclosed herein.
The peptide used as an immune antigen may be the peptide of the present invention or a fragment of the peptide of the present invention. In addition, the peptide may be bound to a carrier or conjugated to a carrier to increase immunogenicity. Keyhole Limpet Hemocyanin (KLH) is known as a carrier. Methods for binding KLH to peptides are also well known in the art.
Any mammal may be immunized with the antigen described above, and compatibility with the parent cell used for cell fusion is preferably considered when producing monoclonal antibodies. Typically, animals of the order Rodentia (Rodentia), lagomorpha (Lagomorpha) or primates (Primate) are used. Rodents include, for example, mice, rats, and hamsters. Animals of the order lagomorpha include, for example, rabbits. Primates include, for example, angustens (old world monkeys) such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys, baboons, and chimpanzees.
Methods of immunizing animals with antigens are known in the art. Intraperitoneal and subcutaneous injection of antigens are standard methods of immunizing mammals. More specifically, the antigen may be diluted and suspended in an appropriate amount of Phosphate Buffered Saline (PBS), physiological saline, or the like. If desired, the antigen suspension may be mixed with an appropriate amount of standard adjuvant such as Freund's (Freund's) complete adjuvant, formed into an emulsion, and then administered to the mammal. Then, it is preferable to use the antigen mixed with an appropriate amount of Freund's incomplete adjuvant for administration several times every 4 to 21 days. Immunization can be performed using a suitable carrier. After immunization as described above, the serum may be assayed for an increase in the amount of desired antibody by standard methods.
Polyclonal antibodies to the peptides of the invention may be prepared by collecting blood from a mammal, which has confirmed an increase in serum levels of the desired antibody after immunization, and separating serum from the blood by any conventional method. The polyclonal antibody may be a serum comprising the polyclonal antibody, or may be isolated from said serum, a fraction comprising the polyclonal antibody. Immunoglobulin G or M may be prepared from a fraction recognizing only the peptide of the present invention using, for example, an affinity column coupled with the peptide of the present invention, and further purifying the fraction using a protein a or protein G column.
To prepare monoclonal antibodies, immune cells are harvested from a mammal and subjected to cell fusion when confirming an increase in serum levels of the desired antibodies after immunization. Immune cells for cell fusion may preferably be obtained from the spleen. For other parent cells fused to the immune cells described above, for example, mammalian myeloma cells may be used, and more preferably myeloma cells that have acquired the drug-selective properties of the fused cells.
The above immune cells may be fused with myeloma cells according to known Methods, for example, the method of MILSTEIN ET al (GALFRE AND MILSTEIN, methods Enzymol,1981, 73:3-46).
For hybridomas obtained by cell fusion, selection can be performed in standard selection media such as HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). Cell culture is typically continued for a sufficient period of time (e.g., days to weeks) in HAT medium to allow all other cells (non-fused cells) except the desired hybridoma to die. Hybridoma cells producing the desired antibody are then selected and cloned by standard limiting dilution (STANDARD LIMITING edition).
In addition to the above-described methods of immunizing a non-human animal with an antigen to prepare hybridomas, human lymphocytes, such as those infected with EB virus, can be immunized in vitro with a peptide, cells expressing the peptide, or a lysate thereof. The immunized lymphocytes can then be fused with an immortalized human-derived myeloma cell, such as U266, to produce the desired hybridoma that produces a human antibody capable of binding to the peptide (JPS 63-17688).
The resulting hybridoma was then transplanted into the abdominal cavity of a mouse, and ascites were extracted. The resulting monoclonal antibodies may be purified, for example, by ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or affinity column coupled with the peptides of the present invention.
Alternatively, immune cells (e.g., immunized lymphocytes) can be produced by immortalizing antibodies with oncogenes and used to prepare monoclonal antibodies.
The monoclonal antibodies thus obtained can also be recombinantly produced using genetic engineering techniques (see, e.g., borrebaeck AND LARRICK, therapeutic Monoclonal Antibodies, published by MACMILLAN PUBLISHERS LTD in the uk (1990)). For example, DNA encoding an antibody may be cloned from an immune cell such as a hybridoma or via an immune lymphocyte, inserted into a suitable vector, and introduced into a host cell to produce a recombinant antibody. The present invention also provides a recombinant antibody prepared as described above.
Furthermore, the antibody of the present invention may be an antibody fragment or a modified antibody as long as it binds to the peptide of the present invention. For example, it is desirable that the antibody fragment contains the antigen binding site of the antibody. Specifically, the antibody fragment may be Fab, F (ab') 2, fv, or single chain Fv (scFv) in which Fv fragments from H and L chains are joined by a suitable linker (Huston et al, proc NATL ACAD SCI USA,1988, 85:5879-83). More specifically, antibody fragments may be generated by treating antibodies with enzymes such as papain or pepsin. Alternatively, a gene encoding an antibody fragment may be constructed, inserted into an expression vector, and expressed in a suitable host cell (see, e.g., Co et al.,J Immunol,1994,152:2968-76;Better and Horwitz,Methods Enzymol,1989,178:476-96;Pluckthun and Skerra,Methods Enzymol,1989,178:497-515;Lamoyi,Methods Enzymol,1986,121:652-63;Rousseaux et al.,Methods Enzymol,1986,121:663-9;Bird and Walker, trends Biotechnol,1991, 9:132-7)).
Antibodies can be modified by conjugation to a variety of molecules such as polyethylene glycol (PEG). The present invention provides antibodies that have been subjected to such modifications. The modified antibodies can be obtained by chemically modifying the antibodies. These modification methods are conventional in the art.
Alternatively, the antibody of the present invention may be obtained in the form of a chimeric antibody between a variable region derived from a non-human antibody and a constant region derived from a human antibody or a humanized antibody comprising Complementarity Determining Regions (CDRs) derived from a non-human antibody, framework Regions (FRs) derived from a human antibody and constant regions. Such antibodies can be prepared according to known techniques. Humanization can be performed by substituting the corresponding sequences of the human antibodies with non-human antibody CDR sequences (see, e.g., verhoeyen et al, science,1988, 239:1534-6). Thus, such humanized antibodies are chimeric antibodies in which a small portion of the human variable domain has been replaced with a corresponding sequence from a non-human species.
Fully human antibodies may also be used, such antibodies comprising human variable regions in addition to human framework and constant regions. Such antibodies can be prepared using a variety of techniques known in the art. For example, in vitro methods include the use of recombinant libraries of human antibody fragments displayed on phage (e.g., hoogenboom & Winter, J. Mol. Biol.,1991,227: 381). Similarly, antibodies can be generated by introducing human immunoglobulin loci into transgenic animals, such as mice that are partially or fully inactivated with endogenous immunoglobulin genes. The method is described, for example, in U.S. Pat. No 6,150,584, 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016.
The antibody obtained as above may be purified to homogeneity. For example, the separation and purification of antibodies can be performed according to the separation and purification methods used for general proteins. For example, the antibody may be isolated and purified by appropriately selecting and using a combination of column chromatography such as affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis and isoelectric focusing (Antibodies: A Laboratory Manual. Ed Harlow and DAVID LANE, cold Spring Harbor Laboratory (1988)), but is not limited thereto. Protein a and protein G columns may be used as affinity columns. Exemplary protein a columns that may be used include, for example, hyper D, POROS, and Sepharose f.f. (Pharmacia).
In addition to affinity chromatography, exemplary chromatography includes, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and the like (STRATEGIES FOR PROTEIN PURIFICATION and Characterization: A Laboratory Course Manual. Ed Daniel R.Marshak et al Cold Spring Harbor Laboratory Press (1996)). Chromatographic procedures such as HPLC and FPLC can be performed by liquid chromatography.
For example, the antigen binding activity of an antibody of the invention can be measured by using absorbance measurement, enzyme-linked immunosorbent assay (ELISA), enzyme Immunoassay (EIA), radioimmunoassay (RIA) and/or Immunofluorescence (IF). In ELISA, the antibodies of the invention are immobilized on a plate, the peptides of the invention are applied to the plate, and then a sample containing the desired antibodies, such as culture supernatant of antibody-producing cells or purified antibodies, is applied. A second antibody, labeled with an enzyme (such as alkaline phosphatase), that recognizes the first antibody is then applied and the plate is incubated. Next, after washing, an enzyme substrate, such as p-nitrophenyl phosphate, is added to the plate and the antigen binding activity of the sample is assessed by measuring absorbance. To evaluate the binding activity of an antibody, a peptide fragment such as a C-terminal or N-terminal fragment may be used as an antigen. BIAcore (Pharmacia) can be used to evaluate the activity of the antibodies of the invention.
The peptides of the present invention using the above method can be detected or measured by exposing the antibodies of the present invention to a sample supposed to contain the peptides of the present invention and detecting or measuring immune complexes formed between the antibodies and the peptides.
For example, the antibodies of the invention can be used to detect the presence of the peptides of the invention in a blood sample (e.g., a serum sample) of a subject. Alternatively, the peptides of the invention may be used to detect the presence of antibodies of the invention in a blood sample (e.g., a serum sample) from a subject. The results of measuring the peptide of the invention or the antibody of the invention in a blood sample of a subject may be used in a subject to select for administration of the pharmaceutical composition of the invention or to monitor the efficacy of the pharmaceutical composition. Furthermore, it has been reported that patients with antibodies against peptides administered as vaccines may have a high responsiveness to the vaccine. Thus, when the peptide of the present invention is administered as a vaccine using the patient's antibody as an index (index), the peptide of the present invention can be used as an immunoassay antigen for selecting patients with high responsiveness.
XII vector and host cell
The present invention provides a vector comprising a polynucleotide encoding the peptide of the present invention and a host cell into which the vector is introduced. The vectors of the invention may be used to retain the polynucleotides of the invention in a host cell, to express the peptides of the invention in a host cell, or to administer the polynucleotides of the invention for gene therapy.
When E.coli is a host cell and a vector is amplified and produced in large amounts in E.coli (e.g., JM109, DH5-alpha, HB101 or XL 1-Blue), the vector needs to have an "origin of replication" for amplification in E.coli and a marker gene (e.g., a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol, etc.) for selection of the transformed E.coli. For example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script and the like can be used. In addition, pGEM-T, pDIRECT and pT7 can be used for cloning and the above vectors. When the vector is used for producing the peptide of the present invention, an expression vector may be used. For example, an expression vector to be expressed in E.coli should have the characteristics described above for amplification in E.coli. When E.coli such as JM109, DH5-alpha, HB101 or XL1-Blue is used as the host cell, the vector should have a promoter capable of efficiently expressing a desired gene in E.coli, for example, the lacZ promoter (Ward et al Nature 341:544-6 (1989); FASEB J6:2422-7 (1989)), the araB promoter (Better et al Science 240:1041-3 (1988)), the T7 promoter and the like. In this regard, pGEX-5X-1 (Pharmacia), "QIAexpress System" (Qiagen), pEGFP and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase) may be used instead of the above vectors. In addition, the vector may also contain a signal sequence for peptide secretion. Exemplary signal sequences directing secretion of the peptide into the periplasm of E.coli are the pelB signal sequences ((Lei et al, J Bacteriol,1987, 169:4379.) means for introducing the vector into a target host cell include, for example, the calcium chloride method and electroporation method.
In addition to E.coli, polypeptides of the invention can be produced using, for example, mammalian-derived expression vectors (e.g., pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res (17): 5322 (1990)), pEF, pCDM 8), insect-cell-derived expression vectors (e.g., the "Bac-to-BAC baculovirus expression system" (GIBCO BRL), pBacPAK 8), plant-derived expression vectors (e.g., pMH1, pMH 2), animal-virus-derived expression vectors (e.g., pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (e.g., pZIpneo), yeast-derived expression vectors (e.g., "Pichia pastoris expression kit" (Invitrogen), pNV, SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608, pKTH 50).
For expression of the vector in animal cells such as CHO, COS or NIH3T3 cells, the vector should have promoters necessary for expression in said cells, for example the SV40 promoter (Mulligan et al, nature,1979, 277:108), the MMLV-LTR promoter, the EF1-alpha promoter (Mizushima et al, nucleic Acids Res,1990, 18:5322), the CMV promoter, etc., and marker genes preferably used for selection of transformants (for example drug resistance genes selected by drugs (e.g. neomycin, G418). Examples of known vectors having these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.
Embodiments of the present invention are exemplified below based on the above description, however, the present invention is not limited to these embodiments.
[1] A peptide of less than 15 amino acids having cytotoxic T Cell (CTL) inducibility comprising an amino acid sequence selected from the group consisting of:
(a) Amino acid sequences ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61 selected from the group consisting of
(B) Amino acid sequences ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61 in which 1, 2 or several amino acids are substituted, deleted, inserted and/or added to the amino acid sequences selected from the following groups.
[2] The peptide of [1] selected from the following groups (i) to (ii):
(i) A peptide comprising an amino acid sequence comprising one or more substitutions selected from the group consisting of SEQ ID NOs 1,2,3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 introduced into an amino acid sequence selected from the group consisting of:
(a) Substitution of the first amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid;
(b) Substitution of the second amino acid from the N-terminus with an amino acid selected from the group consisting of phenylalanine, tyrosine, alanine, isoleucine, leucine and valine, and
(C) Substitution of the C-terminal amino acid with an amino acid selected from the group consisting of arginine and lysine;
(ii) A peptide comprising an amino acid sequence comprising one or more substitutions selected from the group consisting of SEQ ID NOs 48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61 introduced into an amino acid sequence selected from the group consisting of:
(a) Replacing a second amino acid from the N-terminus with an amino acid selected from the group consisting of threonine and serine;
(b) Substitution of the third amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid, and
(C) The C-terminal amino acid was replaced with tyrosine.
[3] The peptide of claim 1, consisting of an amino acid sequence selected from the group consisting of ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61.
[4] A polynucleotide encoding the peptide of any one of [1] to [3 ].
[5] A composition comprising a pharmaceutically acceptable carrier and at least one ingredient selected from the following groups (a) to (e):
(a) One or more types of the peptide of any one of [1] to [3 ];
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of [1] to [3 ];
(c) An Antigen Presenting Cell (APC) that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on its cell surface;
(d) Exosomes presenting complexes of the peptide of any one of [1] to [3] with HLA antigens on the cell surface thereof, and
(E) A CTL targeting the peptide of any one of [1] to [3 ].
[6] The composition of [5], which is a composition for inducing CTL, wherein the ingredient is at least one ingredient selected from the following groups (a) to (d):
(a) One or more types of the peptide of any one of [1] to [3 ];
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of [1] to [3 ];
(c) An Antigen Presenting Cell (APC) which presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on its cell surface, and
(D) Exosomes presenting complexes of the peptide of any one of [1] to [3] with HLA antigens on their cell surfaces.
[7] The composition of [5], which is a pharmaceutical composition.
[8] The composition of [7] for use in one or more applications selected from the group consisting of (i) cancer treatment, (ii) cancer prevention (prophltaxis) and (iii) prevention (prevention) of postoperative cancer recurrence.
[9] The composition of [7] for inducing an immune response against cancer.
[10] The composition of [8] or [9], wherein the cancer is selected from the group consisting of Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, and testicular tumor.
[11] The composition of any one of [5 to [10], formulated for administration to at least one HLA-positive subject selected from the group consisting of HLA-A33 and HLA-A01.
[12] A method of inducing APC having CTL inducibility comprising the step of:
(a) Contacting APC with the peptide of any one of [1] to [3] in vitro, ex vivo or in vivo, and
(B) Introducing a polynucleotide encoding the peptide of any one of [1] to [3] into APC.
[13] A method of inducing CTLs comprising the steps selected from the group consisting of (a) to (c) below:
(a) Co-culturing CD8 positive T cells with APCs that present on their surface complexes of HLA antigens with peptides of any one of [1] to [3 ];
(b) Co-culturing CD8 positive T cells with exosomes presenting complexes of HLA antigens on their surfaces with peptides of any one of [1] to [3 ];
(c) Introducing a polynucleotide encoding each subunit of a T Cell Receptor (TCR) capable of binding to a peptide of any one of [1] to [3] presented on the cell surface by an HLA antigen into a CD8 positive T cell.
[14] An APC presenting on its surface a complex of an HLA antigen and a peptide of any one of [1] to [3 ].
[15] The APC of [14], which is induced by the method of [12 ].
[16] A CTL targeting the peptide of any one of [1] to [3 ].
[17] The CTL of [16], which is induced by the method of [13 ].
18. A method of inducing an immune response against cancer comprising administering to a subject at least one component selected from the group consisting of (a) to (e):
(a) One or more types of the peptide of any one of [1] to [3 ];
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of [1] to [3 ];
(c) An APC which presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on its cell surface;
(d) Exosomes presenting complexes of the peptide of any one of [1] to [3] with HLA antigens on the cell surface thereof, and
(E) A CTL targeting the peptide of any one of [1] to [3 ].
19. A method of treating and/or preventing cancer, and/or preventing postoperative recurrence thereof, the method comprising administering to a subject at least one ingredient selected from the group consisting of (a) to (e):
(a) One or more types of the peptide of any one of [1] to [3 ];
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of [1] to [3 ];
(c) An APC which presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on its cell surface;
(d) Exosomes presenting complexes of the peptide of any one of [1] to [3] with HLA antigens on the cell surface thereof, and
(E) A CTL targeting the peptide of any one of [1] to [3 ].
[20] An antibody that binds to the peptide of any one of [1] to [3 ].
[21] A method of screening for peptides having CTL inducibility, comprising the steps of:
(a) Generating a candidate sequence consisting of an amino acid sequence, wherein one, two or several amino acid residues ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61 are substituted, deleted, inserted and/or added to the original amino acid sequence consisting of an amino acid sequence selected from the group consisting of;
(b) Selecting a candidate sequence having no significant homology (sequence identity) with any known human gene product other than FOXM1 from the candidate sequences generated in (a);
(c) Contacting the APC with a peptide consisting of the candidate sequence selected in (b);
(d) Contacting the APC of (c) with a CD 8-positive T cell, and
(E) Peptides having equal or higher CTL inducibility than peptides consisting of the original amino acid sequence were selected.
[22] Use of at least one member selected from the group consisting of the following (a) to (e) for the preparation of a composition for inducing an immune response against cancer:
(a) One or more types of the peptide of any one of [1] to [3 ];
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of [1] to [3 ];
(c) An Antigen Presenting Cell (APC) that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;
(d) Exosomes presenting complexes of the peptide of any one of [1] to [3] with HLA antigens on the cell surface, and
(E) A CTL targeting the peptide of any one of [1] to [3 ].
[23] Use of at least one ingredient selected from the following groups (a) to (e) for the preparation of a pharmaceutical composition for the treatment and/or prevention of cancer and/or the prevention of postoperative recurrence thereof:
(a) One or more types of the peptide of any one of [1] to [3 ];
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of [1] to [3 ];
(c) An Antigen Presenting Cell (APC) that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;
(d) Exosomes presenting complexes of the peptide of any one of [1] to [3] with HLA antigens on the cell surface, and
(E) A CTL targeting the peptide of any one of [1] to [3 ].
[24] Use of at least one member selected from the group consisting of the following (a) to (e) for inducing an immune response against cancer:
(a) One or more types of the peptide of any one of [1] to [3 ];
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of [1] to [3 ];
(c) An Antigen Presenting Cell (APC) that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;
(d) Exosomes presenting complexes of the peptide of any one of [1] to [3] with HLA antigens on the cell surface, and
(E) A CTL targeting the peptide of any one of [1] to [3 ].
[25] Use of at least one component selected from the group consisting of the following (a) to (e) for the treatment and/or prevention of cancer and/or for the prevention of postoperative recurrence thereof:
(a) One or more types of the peptide of any one of [1] to [3 ];
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of [1] to [3 ];
(c) An Antigen Presenting Cell (APC) that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;
(d) Exosomes presenting complexes of the peptide of any one of [1] to [3] with HLA antigens on the cell surface, and
(E) A CTL targeting the peptide of any one of [1] to [3 ].
[26] A method of inducing cytotoxic activity against FOXM 1-expressing cells, comprising the step of administering at least one component selected from the following groups (a) to (e) to a subject:
(a) One or more types of the peptide of any one of [1] to [3 ];
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of [1] to [3 ];
(c) An Antigen Presenting Cell (APC) that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;
(d) Exosomes presenting complexes of the peptide of any one of [1] to [3] with HLA antigens on the cell surface, and
(E) A CTL targeting the peptide of any one of [1] to [3 ].
[27] A lyophilized formulation comprising one or more types of peptide of any one of [1] to [3 ].
[28] A pharmaceutical composition prepared by a method comprising dissolving one or more types of the peptide of any one of [1] to [3] in a water-soluble carrier, and performing filtration sterilization.
[29] A filter sterilized aqueous solution which is an aqueous solution comprising one or more types of the peptide of any one of [1] to [3] and a water-soluble carrier.
[30] An emulsion comprising one or more types of the peptide of any one of [1] to [3], a water-soluble carrier and an oil adjuvant.
[31] A kit comprising a container containing the pharmaceutical composition of any one of [5] to [11], and a container containing an adjuvant.
[32] A kit comprising a container storing a lyophilized formulation comprising the peptide of any one of [1] to [3], a container storing an adjuvant, and a container storing the lyophilized formulation in a solution.
The invention is herein explained in detail with reference to specific embodiments thereof. It should be understood, however, that the foregoing description is illustrative and explanatory thereof in nature, and is intended to illustrate the invention and its preferred embodiments. Those skilled in the art will readily recognize from such routine experimentation that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Accordingly, the invention is not to be limited by the foregoing description but is intended to be defined by the appended claims and their equivalents.
Hereinafter, the present invention will be described in more detail with reference to examples. However, while the following materials, methods, and examples may be used to assist one of ordinary skill in making and using certain embodiments of the present invention, these are intended merely to illustrate various aspects of the present invention and thus are in no way limiting of the scope of the present invention. As one of ordinary skill in the art will readily recognize, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
All prior art documents cited herein are incorporated by reference into the present specification.
Examples
Example 1
Materials and methods
Cell lines
C1R cells (HLA-A negative and HLA-B negative human B lymphoblastic cell lines), and COS7 cells (African green monkey kidney cell lines) were purchased from ATCC.
Generation of target cells with stable HLA-A 33:03 expression
C1R (C1R-A33) stably expressing HLA-A 33:03 was used as cells for stimulating CTL. cDNA encoding HLA-A 33:03 was amplified by PCR and cloned into an expression vector. Under drug selection, C1R cells introduced with HLA-A 33:03 gene expression vector were cultured for two weeks in medium containing G418 (Invitrogen). The G418 resistant C1R cell suspension was diluted, seeded in 96-well plates, and further selectively cultured in a medium containing G418 for 30 days. HLA-A 33:03 expression in C1R cells was confirmed by flow cytometry analysis.
Selection of FOXM 1-derived peptides
FOXM 1-derived 9-mer and 10-mer peptides that were expected to bind HLA-A x 33:03 molecules were determined using a binding prediction server "NetMHC pan2.8"(www.cbs.dtu.dk/services/NetMHCpan-2.8/)(Buus et al.,Tissue Antigens.2003,62(5):378-84;Nielsen et al.,Protein Sci.2003,12(5):1007-17;Bioinformatics.2004,20(9):1388-97).
Peptide synthesis
The peptides were synthesized by AMERICAN PEPTIDE Company (Sunnyvale, CA) according to standard solid phase synthesis and purified by reverse phase High Performance Liquid Chromatography (HPLC). The quality (purity 90% or higher) of the peptide was ensured by HPLC and mass spectrometry. The peptide was dissolved in dimethyl sulfoxide (final concentration: 20 mg/ml) and stored at-80 ℃
In vitro CTL induction
Monocyte-derived Dendritic Cells (DCs) are used as antigen presenting cells to induce specific Cytotoxic T Lymphocyte (CTL) responses against peptides presented on Human Leukocyte Antigens (HLA). As reported in the literature, DCs are generated in vitro (NAKAHARA S ET al, CANCER RES 2003,63 (14): 4112-8). Specifically, peripheral Blood Mononuclear Cells (PBMCs) collected from healthy volunteers with Ficoll-Paque plus solution (Pharmacia) were inoculated into plastic tissue culture dishes (Corning) to adhere the mononuclear cells in the PBMCs to the dishes. This was incubated in the presence of 1000IU/ml granulocyte macrophage colony stimulating factor (R & D System) and 1000IU/ml Interleukin (IL) -4 (R & D System) for 7 days. AIM-V medium (Invitrogen) containing 5% inactivated AB type serum (ABS) was used as the medium. DC induced differentiation from monocytes using cytokines was pulsed with 20 μg/ml of each synthetic peptide (37 degrees Celsius, 3 hours). Peptide impact was performed in AIM-V medium containing 3. Mu.g/ml beta 2-microglobulin. These peptide-pulsed DCs were inactivated by X-ray irradiation (20 Gy), mixed with autologous CD8 positive T cells obtained by using CD8 positive isolation kit (Invitrogen) at a 1:20 ratio (1.5x10 4 DCs and 3X10 5 CD8 positive T cells), and cultured in 48 well plates (Corning). Each well contained 0.5ml of 5% ABS/AIM-V medium, and IL-7 (R & D System) (final concentration: 10 ng/ml) was added thereto. Two days after the start of the culture, IL-2 (Novartis) (final concentration: 20 IU/ml) was added. On days 7 and 14 of culture, CD8 positive T cells were further stimulated with peptide-pulsed DCs. The DCs were prepared in use by the same method as described above. After 21 days (after 3 DC stimulations), IFN-gamma production was confirmed for peptide-shocked C1R-A33 using a human Interferon (IFN) -gamma enzyme-linked immunospot (ELISPOT) assay (Tanaka H et al.,Br J Cancer 2001,84(1):94-9;Umano Y et al.,Br J Cancer 2001,84(8):1052-7;Uchida N et al.,Clin Cancer Res 2004,10(24):8577-86;Suda T et al.,Cancer Sci 2006,97(5):411-9;Watanabe T et al.,Cancer Sci 2005,96(8):498-506).
CTL amplification program
CTL was amplified in culture using a method similar to that reported by Riddell et al (WALTER EA ET al, N Engl J Med 1995,333 (16): 1038-44;Riddell SR et al, nat Med 1996,2 (2): 216-23). CTLs were cultured with two types of mitomycin C-treated human B lymphoblastic cell lines (5X 10 6 cells per 25ml of each medium) and anti-CD 3 antibodies (final concentration 40 ng/ml) in 25ml of 5% ABS/AIM-V medium. One day after the start of culture, IL-2 (final concentration: 120 IU/ml) was added to the culture. On days 5, 8 and 11, the medium was replaced with 5% ABS/AIM-V medium containing IL-2 (final concentration: 30 IU/ml) (Tanaka H et al.,Br J Cancer 2001,84(1):94-9;Umano Y et al.,Br J Cancer 2001,84(8):1052-7;Uchida N et al.,Clin Cancer Res 2004,10(24):8577-86;Suda T et al.,Cancer Sci 2006,97(5):411-9;Watanabe T et al.,Cancer Sci 2005,96(8):498-506).
Establishment of CTL clones
After in vitro induction of CTL, CTL were seeded at 1 cell/well or 10 cells/well onto 96-well round-bottomed microplates (Nalge Nunc International). In AIM-V/5% ABS/AIM-V medium containing a total of 150. Mu.l/well of anti-CD 3 antibody (final concentration: 30 ng/ml) and IL-2 (final concentration: 125 IU/ml), CTLs were co-cultured with two types of mitomycin C treated human B lymphoblastic cell lines (1X 10 4 cells/well). After 10 days, 50. Mu.l of 5% ABS/AIM-V containing 500IU/ml IL-2 was added to the culture. Amplification of the peptides exhibiting specific IFN-gamma production in the ELISPOT assay on day 14 or after using the same procedure as described above CTL(Uchida N et al.,Clin Cancer Res 2004,10(24):8577-86;Suda T et al.,Cancer Sci 2006,97(5):411-9;Watanabe T et al.,Cancer Sci 2005,96(8):498-506).
Validation of IFN-gamma production
To confirm peptide-specific IFN-gamma production of CTLs induced by peptides, IFN-gamma ELISPOT assays and IFN-GAMMA ELISA were performed. Peptide-shocked C1R-A33 (1X 10 4 cells/well) was prepared as target cells. IFN-gamma ELISPOT assays and IFN-GAMMA ELISA were performed according to the manufacturer's instructions.
Preparation of cells that forced expression of FOXM1 Gene and HLA-A 33:03
CDNA encoding FOXM1 or HLA-A 33:03 genes was amplified by PCR. The PCR amplification products were each cloned into an expression vector. One or both of the FOXM1 gene expression vector and HLA-A 33:03 gene expression vector were introduced into COS7 cells (which are HLa negative cell lines) using Lipofectamine 2000 (Invitrogen). One day after gene introduction, COS7 cells were dissociated and harvested using Versene (Invitrogen) and used as target cells (5X 10 4 cells/well) to confirm IFN-gamma production.
Results
Prediction of FOXM 1-derived HLA-A 33:03 binding peptide
Tables 1a and 1b show, in decreasing order of binding affinity, the FOXM 1-derived 9-mer peptide and 10-mer peptide that were predicted to bind HLA-A 33:03 by "NETMHC PAN 2.8". A total of 47 peptides potentially having HLA-A 33:03 binding capacity were used as ectopeptide candidates.
TABLE 1a
HLA-A 33:03 binding 9 mer peptide derived from FOXM1
Initial position Amino acid sequence Kd(nM) SEQ ID NO
180 SLSNIQWLR 13.57 1
308 WTIHPSANR 34.66 2
693 QVSGLAANR 75.29 3
190 MSSDGLGSR 132.48 4
515 MLVIQHRER 138.98 5
516 LVIQHRERR 156.26 6
146 AARDVNLPR 230.20 7
140 TLGPKPAAR 248.65 8
389 SLMSSELAR 262.06 9
246 QFAINSTER 277.30 10
289 LSLHDMFVR 439.26 11
228 SWQNSVSER 481.46 12
125 TQTSYDAKR 519.42 13
489 DSSQSPTPR 646.55 14
270 FPYFKHIAK 766.69 15
216 QVKVEEPSR 835.12 16
502 YSGLRSPTR 855.62 17
321 DQVFKQQKR 1343.52 18
393 SELARHSKR 1443.91 19
341 KTELPLGAR 1574.92 20
547 FSEGPSTSR 1679.70 21
The starting position indicates the number of amino acid residues from the N-terminus of FOXM 1.
The dissociation constant [ Kd (nM) ] is derived from "NetMHC2.8".
TABLE 1b
HLA-A 33:03 binding 10-mer peptide derived from FOXM1
Initial position Amino acid sequence Kd(nM) SEQ ID NO
514 EMLVIQHRER 26.71 22
139 ETLGPKPAAR 33.00 23
179 NSLSNIQWLR 34.89 24
288 NLSLHDMFVR 91.77 25
501 SYSGLRSPTR 109.92 26
515 MLVIQHRERR 114.82 27
307 FWTIHPSANR 131.76 28
398 HSKRVRIAPK 150.52 29
245 IQFAINSTER 172.14 30
269 HFPYFKHIAK 176.26 31
124 QTQTSYDAKR 190.55 32
595 STPSKSVLPR 240.63 33
394 ELARHSKRVR 278.30 34
247 FAINSTERKR 299.53 35
546 LFSEGPSTSR 332.38 36
392 SSELARHSKR 608.20 37
227 ASWQNSVSER 753.17 38
391 MSSELARHSK 875.46 39
326 QQKRPNPELR 880.19 40
607 ESWRLTPPAK 1179.88 41
265 WIEDHFPYFK 1214.75 42
497 RPKKSYSGLR 1332.87 43
471 EWPSPAPSFK 1418.64 44
4 SPRRPLILKR 1427.21 45
388 ASLMSSELAR 1586.13 46
651 SAPPLESPQR 1587.27 47
The starting position indicates the number of amino acid residues from the N-terminus of FOXM 1.
The dissociation constant [ Kd (nM) ] is derived from "NetMHC2.8".
CTL induction by predicted FOXM 1-derived HLA-A 33:03 restriction peptides
FOXM 1-derived peptide-specific CTLs were induced according to the protocol described in "materials and methods". Peptide-specific IFN-gamma production was confirmed by ELISPOT assay (FIG. 1). Peptide-specific IFN-gamma production was observed in the following wells:
In well #6 (a) using OXM1-A33-9-180 (SEQ ID NO: 1)
In well #3 (b) using FOXM1-A33-9-308 (SEQ ID NO: 2)
In well #4 (c) using FOXM1-A33-9-693 (SEQ ID NO: 3)
In well #3 (d) using FOXM1-A33-9-516 (SEQ ID NO: 6)
In well #5 (e) using FOXM1-A33-9-146 (SEQ ID NO: 7)
In well #6 (f) using FOXM1-A33-9-289 (SEQ ID NO: 11)
In well #6 (g) using FOXM1-A33-9-228 (SEQ ID NO: 12)
In well #4 (h) using FOXM1-A33-9-502 (SEQ ID NO: 17)
In well #2 (i) using FOXM1-A33-9-321 (SEQ ID NO: 18)
In well #6 (j) using FOXM1-A33-9-341 (SEQ ID NO: 20)
In well #8 (k) using FOXM1-A33-10-514 (SEQ ID NO: 22)
In well #6 (l) using FOXM1-A33-10-179 (SEQ ID NO: 24)
In well #5 (m) using FOXM1-A33-10-501 (SEQ ID NO: 26)
In well #5 (n) using FOXM1-A33-10-124 (SEQ ID NO: 32)
In well #3 (o) using FOXM1-A33-10-595 (SEQ ID NO: 33)
In well #5 (p) using FOXM1-A33-10-546 (SEQ ID NO: 36)
In well #6 (q) using FOXM1-A33-10-391 (SEQ ID NO: 39)
In well #3 (r) using FOXM1-A33-10-607 (SEQ ID NO: 41)
In well # 2(s) using FOXM1-A33-10-265 (SEQ ID NO: 42)
In well #6 (t) and using FOXM1-A33-10-4 (SEQ ID NO: 45)
In well #5 (u) using FOXM1-A33-10-388 (SEQ ID NO: 46).
Meanwhile, no specific IFN-gamma production was observed for the other peptides shown in tables 1a and 1 b. For example, NO specific IFN-gamma production (v) was observed for FOXM1-A33-10-288 (SEQ ID NO: 25). As a result, although all peptides had the potential to bind HLA-A 33:03, 21 peptides were selected as peptides with CTL-inducing ability.
Establishment of CTL lines and clones specific for HLA-A 33:03 restricted FOXM 1-derived peptides
CTL lines were established by proliferating cells in wells #3 using FOXM1-A33-9-308 (SEQ ID NO: 2), in well #5 using FOXM1-A33-9-146 (SEQ ID NO: 7), in well #6 using FOXM1-A33-10-391 (SEQ ID NO: 39) (c), and in well #6 using FOXM1-A33-10-265 (SEQ ID NO: 42) in IFN-gamma ELISPOT assays. As a result of measurement of IFN-gamma by ELISA, it was observed that IFN-gamma was produced by CTL lines against target cells (C1R-A33) (FIG. 2) target cells (a) impacted with well #3 having FOXM1-A33-9-308 (SEQ ID NO: 2), target cells (b) impacted with well #5 having FOXM1-A33-9-146 (SEQ ID NO: 7), target cells (C) impacted with well #6 having FOXM1-A33-10-391 (SEQ ID NO: 39), or FOXM1-A33-10-265 (SEQ ID NO: 42). Furthermore, CTL clones were established by limiting dilution from CTL lines as described in the materials and methods section above. As a result of measurement of IFN-gamma by ELISA, CTL clones stimulated with either FOXM1-A33-9-308 (SEQ ID NO: 2) (a) or FOXM1-A33-9-146 (SEQ ID NO: 7) (b) each showed peptide-specific IFN-gamma production (FIG. 3).
IFN-gamma production against target cells expressing FOXM1 and HLA-A 33:03
IFN-gamma production was studied for specific CTL clones of FOXM1-A33-9-308 (SEQ ID NO: 2) against target cells expressing FOXM1 and HLA-A 33:03. COS7 cells expressing both FOXM1 and HLA-A 33:03 were prepared as target cells. COS7 cells expressing one of FOXM1 and HLA-A 33:03 were prepared as negative control cells. FOXM1-A33-9-308 (SEQ ID NO: 2) specific CTL clones showed IFN-gamma production against COS7 cells expressing both FOXM1 and HLA-A 33:03 (FIG. 4). On the other hand, no significant IFN-gamma production was observed against the negative control cells. This clearly demonstrates that FOXM1-a33-9-308 (SEQ ID NO: 2) is a peptide produced by antigen processing and is presented on the cell surface with HLA-A 33:03 molecules and recognized by CTLs. The results indicate that FOXM1-a33-9-308 (SEQ ID NO: 2) can be useful as a cancer vaccine for patients whose FOXM1 expression in cancer cells is enhanced.
Homology analysis of antigenic peptides
FOXM1-A33-9-180(SEQ ID NO:1)、FOXM1-A33-9-308(SEQ ID NO:2)、FOXM1-A33-9-693(SEQ ID NO:3)、FOXM1-A33-9-516(SEQ ID NO:6)、FOXM1-A33-9-146(SEQ ID NO:7)、FOXM1-A33-9-289(SEQ ID NO:11)、FOXM1-A33-9-228(SEQ ID NO:12)、FOXM1-A33-9-502(SEQ ID NO:17)、FOXM1-A33-9-321(SEQ ID NO:18)、FOXM1-A33-9-341(SEQ ID NO:20)、FOXM1-A33-10-514(SEQ ID NO:22)、FOXM1-A33-10-179(SEQ ID NO:24)、FOXM1-A33-10-501(SEQ ID NO:26)、FOXM1-A33-10-124(SEQ ID NO:32)、FOXM1-A33-10-595(SEQ ID NO:33)、FOXM1-A33-10-546(SEQ ID NO:36)、FOXM1-A33-10-391(SEQ ID NO:39)、FOXM1-A33-10-607(SEQ ID NO:41)、FOXM1-A33-10-265(SEQ ID NO:42)、FOXM1-A33-10-4(SEQ ID NO:45) And FOXM1-A33-10-388 (SEQ ID NO: 46) have been shown to induce CTLs that show peptide-specific IFN-gamma production. Thus, to confirm that FOXM1-A33-9-180(SEQ ID NO:1)、FOXM1-A33-9-308(SEQ ID NO:2)、FOXM1-A33-9-693(SEQ ID NO:3)、FOXM1-A33-9-516(SEQ ID NO:6)、FOXM1-A33-9-146(SEQ ID NO:7)、FOXM1-A33-9-289(SEQ ID NO:11)、FOXM1-A33-9-228(SEQ ID NO:12)、FOXM1-A33-9-502(SEQ ID NO:17)、FOXM1-A33-9-321(SEQ ID NO:18)、FOXM1-A33-9-341(SEQ ID NO:20)、FOXM1-A33-10-514(SEQ ID NO:22)、FOXM1-A33-10-179(SEQ ID NO:24)、FOXM1-A33-10-501(SEQ ID NO:26)、FOXM1-A33-10-124(SEQ ID NO:32)、FOXM1-A33-10-595(SEQ ID NO:33)、FOXM1-A33-10-546(SEQ ID NO:36)、FOXM1-A33-10-391(SEQ ID NO:39)、FOXM1-A33-10-607(SEQ ID NO:41)、FOXM1-A33-10-265(SEQ ID NO:42)、FOXM1-A33-10-4(SEQ ID NO:45) and FOXM1-A33-10-388 (SEQ ID NO: 46) sequences were derived from FOXM1 only, homology analysis of peptide sequences was performed using the BLAST algorithm (BLAST. Ncbi. Nlm. Nih. Gov/BLAST. Cgi). As a result, FOXM1-A33-9-180(SEQ ID NO:1)、FOXM1-A33-9-308(SEQ ID NO:2)、FOXM1-A33-9-693(SEQ ID NO:3)、FOXM1-A33-9-516(SEQ ID NO:6)、FOXM1-A33-9-146(SEQ ID NO:7)、FOXM1-A33-9-289(SEQ ID NO:11)、FOXM1-A33-9-228(SEQ ID NO:12)、FOXM1-A33-9-502(SEQ ID NO:17)、FOXM1-A33-9-321(SEQ ID NO:18)、FOXM1-A33-9-341(SEQ ID NO:20)、FOXM1-A33-10-514(SEQ ID NO:22)、FOXM1-A33-10-179(SEQ ID NO:24)、FOXM1-A33-10-501(SEQ ID NO:26)、FOXM1-A33-10-124(SEQ ID NO:32)、FOXM1-A33-10-595(SEQ ID NO:33)、FOXM1-A33-10-546(SEQ ID NO:36)、FOXM1-A33-10-391(SEQ ID NO:39)、FOXM1-A33-10-607(SEQ ID NO:41)、FOXM1-A33-10-265(SEQ ID NO:42)、FOXM1-A33-10-4(SEQ ID NO:45) and FOXM1-A33-10-388 (SEQ ID NO: 46) sequences were found only in FOXM 1. Thus, to the knowledge of the present inventors, these peptides are specific for FOXM1, making it almost impossible for these peptides to elicit an undesired immune response against molecules other than FOXM1, which are known to sensitize the human immune system. In summary, novel FOXM 1-derived HLA-A x 33:03 restriction epitope peptides were identified. FOXM 1-derived epitope peptides have been demonstrated to be suitable for cancer immunotherapy.
Example 2
Materials and methods
Cell lines
C1R cells (HLA-A negative and HLA-B negative human B lymphoblastic cell lines), and COS7 cells (African green monkey kidney cell lines) were purchased from ATCC.
Generation of target cells with stable HLA-A 01:01 expression
C1R (C1R-A01) stably expressing HLA-A.times.01:01 was used as cells for stimulating CTL. cDNA encoding HLA-A 01:01 was amplified by PCR and cloned into an expression vector. Under drug selection, C1R cells introduced with HLA-A 01:01 gene expression vector were cultured for two weeks in medium containing G418 (Invitrogen). The G418 resistant C1R cell suspension was diluted, seeded in 96-well plates, and further selectively cultured in a medium containing G418 for 30 days. HLA-A 01:01 expression in C1R cells was confirmed by flow cytometry analysis.
Selection of FOXM 1-derived peptides
FOXM 1-derived 9-mer and 10-mer peptides that were expected to bind HLA-A x 01:01 molecules were determined using a binding prediction server "NetMHC 3.4"(www.cbs.dtu.dk/services/NetMHC-3.4/)(Buus S et al.,Tissue Antigens.2003,62(5):378-84;Nielsen M et al.,Protein Sci.2003,12(5):1007-17;Nielsen M et al.,Bioinformatics.2004,20(9):1388-97).
Peptide synthesis
The peptides were synthesized by AMERICAN PEPTIDE Company (Sunnyvale, CA) according to standard solid phase synthesis and purified by reverse phase High Performance Liquid Chromatography (HPLC). The quality (purity 90% or higher) of the peptide was ensured by HPLC and mass spectrometry. The peptide was dissolved in dimethyl sulfoxide (final concentration: 20 mg/ml) and stored at-80 ℃
In vitro CTL induction
Monocyte-derived Dendritic Cells (DCs) are used as antigen presenting cells to induce specific Cytotoxic T Lymphocyte (CTL) responses against peptides presented on Human Leukocyte Antigens (HLA). As reported in the literature, DCs are generated in vitro (NAKAHARA S ET al, CANCER RES 2003,63 (14): 4112-8). Specifically, peripheral Blood Mononuclear Cells (PBMCs) collected from healthy volunteers (HLA-A 01:01 positive) were inoculated into plastic tissue culture dishes (Corning) to adhere monocytes in the PBMCs to the dishes. This was incubated in the presence of 1000IU/ml granulocyte macrophage colony stimulating factor (R & D System) and 1000IU/ml Interleukin (IL) -4 (R & D System) for 7 days. AIM-V medium (Invitrogen) containing 5% inactivated AB type serum (ABS) was used as the medium. DC induced differentiation from monocytes using cytokines was pulsed with 20 μg/ml of each synthetic peptide (37 degrees Celsius, 3 hours). Peptide impact was performed in AIM-V medium containing 3. Mu.g/ml beta 2-microglobulin. These peptide-pulsed DCs were inactivated by X-ray irradiation (20 Gy), mixed with autologous CD8 positive T cells obtained by using CD8 positive isolation kit (Invitrogen) at a 1:20 ratio (1.5x10 4 DCs and 3X10 5 CD8 positive T cells), and cultured in 48 well plates (Corning). Each well contained 0.5ml of 5% ABS/AIM-V medium, and IL-7 (R & D System) (final concentration: 10 ng/ml) was added thereto. Two days after the start of the culture, IL-2 (Novartis) (final concentration: 20 IU/ml) was added. On days 7 and 14 of culture, CD8 positive T cells were further stimulated with peptide-pulsed DCs. The DCs were prepared in use by the same method as described above. After 21 days (after 3 DC stimulations), IFN-gamma production was confirmed for peptide-shocked C1R-A01 using a human Interferon (IFN) -gamma enzyme-linked immunospot (ELISPOT) assay (Tanaka H et al.,Br J Cancer 2001,84(1):94-9;Umano Y et al.,Br J Cancer 2001,84(8):1052-7;Uchida N et al.,Clin Cancer Res 2004,10(24):8577-86;Suda T et al.,Cancer Sci 2006,97(5):411-9;Watanabe T et al.,Cancer Sci 2005,96(8):498-506).
CTL amplification program
CTL was amplified using a method similar to that disclosed in Riddell et al (WALTER EA ET al, N Engl J Med 1995,333 (16): 1038-44;Riddell SR et al, nat Med 1996,2 (2): 216-23). CTLs were cultured with two types of mitomycin C-treated human B lymphoblastic cell lines (5X 10 6 cells per 25ml of each medium) and anti-CD 3 antibodies (final concentration 40 ng/ml) in 25ml of 5% ABS/AIM-V medium. One day after the start of culture, IL-2 (final concentration: 120 IU/ml) was added to the culture. On days 5, 8 and 11, the medium was replaced with 5% ABS/AIM-V medium containing IL-2 (final concentration: 30 IU/ml) (Tanaka H et al.,Br J Cancer 2001,84(1):94-9;Umano Y et al.,Br J Cancer 2001,84(8):1052-7;Uchida N et al.,Clin Cancer Res 2004,10(24):8577-86;Suda T et al.,Cancer Sci 2006,97(5):411-9;Watanabe T et al.,Cancer Sci 2005,96(8):498-506).
Establishment of CTL clones
After in vitro induction of CTL, CTL were seeded at 1 cell/well or 10 cells/well onto 96-well round-bottomed microplates (Nalge Nunc International). In AIM-V/5% ABS/AIM-V medium containing a total of 150. Mu.l/well of anti-CD 3 antibody (final concentration: 30 ng/ml) and IL-2 (final concentration: 125 IU/ml), CTLs were co-cultured with two types of mitomycin C treated human B lymphoblastic cell lines (1X 10 4 cells/well). After 10 days, 50. Mu.l of 5% ABS/AIM-V containing 500IU/ml IL-2 was added to the culture. Amplification of the peptides exhibiting specific IFN-gamma production in the ELISPOT assay on day 14 or after using the same procedure as described above CTL(Uchida N et al.,Clin Cancer Res 2004,10(24):8577-86;Suda T et al.,Cancer Sci 2006,97(5):411-9;Watanabe T et al.,Cancer Sci 2005,96(8):498-506).
Validation of IFN-gamma production
To confirm the production of peptide-specific IFN-gamm by peptide-induced CTLs, IFN-gamma ELISPOT assays and IFN-GAMMA ELISA were performed. Peptide-shocked C1R-A01 (1X 10 4 cells/well) was prepared as target cells. IFN-gamma ELISPOT assays and IFN-GAMMA ELISA were performed according to the manufacturer's instructions.
Preparation of cells that forced expression of FOXM1 Gene and HLA-A 01:01
CDNA encoding FOXM1 or HLA-A 01:01 genes was amplified by PCR. The PCR amplification products are each integrated into an expression vector. One or both of the FOXM1 gene expression vector and HLA-A 01:01 gene expression vector were introduced into COS7 cells (which are HLa negative cell lines) using Lipofectamine 2000 (Invitrogen). One day after gene introduction, COS7 cells were dissociated and harvested using Versene (Invitrogen) and used as target cells (5X 10 4 cells/well) to confirm IFN-gamma production.
Results
Prediction of FOXM 1-derived HLA-A 01:01 binding peptide
Tables 2a and 2b show 9-mer and 10-mer peptides derived from FOXM1 by "NetMHC 3.4" predicting binding HLA-A 01:01 in descending order of binding affinity. A total of 16 peptides potentially having HLA-A 01:01 binding capacity were used as ectopeptide candidates.
TABLE 2a
Predicting FOXM 1-derived 9-mer peptides that bind HLA-A 01:01
Initial position Amino acid sequence Kd(nM) SEQ ID NO
233 VSERPPYSY 73 48
539 CVDEPELLF 666 49
631 ASDPLPDPL 1807 50
703 LTEGLVLDT 6665 51
231 NSVSERPPY 6867 52
663 SSEPLDLIS 9729 53
392 SSELARHSK 18415 54
494 PTPRPKKSY 19761 55
341 KTELPLGAR 20899 20
The numbering of the starting positions indicates the number of amino acids from the N-terminus of FOXM1 protein corresponding to the first amino acid of the peptide.
The dissociation constant [ Kd (nM) ] was calculated using "NetMHC 3.4".
TABLE 2b
Predicting FOXM 1-derived 10-mer peptides that bind HLA-A 01:01
Initial position Amino acid sequence Kd(nM) SEQ ID NO
566 SSDPASQLSY 15 56
263 YTWIEDHFPY 156 57
308 WTIHPSANRY 554 58
232 SVSERPPYSY 1482 59
663 SSEPlDLISV 10539 60
265 WIEDHFPYFK 18243 42
341 KTELPLGARR 26951 61
The numbering of the starting positions indicates the number of amino acids from the N-terminus of FOXM1 protein corresponding to the first amino acid of the peptide.
The dissociation constant [ Kd (nM) ] was calculated using "NetMHC 3.4".
CTL induction by predicted FOXM 1-derived HLA-A 01:01 restriction peptides
FOXM 1-derived peptide-specific CTLs were induced according to the protocol described in "materials and methods". Peptide-specific IFN-gamma production was confirmed by ELISPOT assay (FIG. 5). Peptide-specific IFN-gamma production was observed in the following wells:
in well #3 using FOXM1-A01-9-233 (SEQ ID NO: 48) (a),
In well #3 using FOXM1-A01-9-539 (SEQ ID NO: 49) (b),
In well #3 with FOXM1-A01-9-631 (SEQ ID NO: 50) (c),
In well #2 using FOXM1-A01-9-231 (SEQ ID NO: 52) (d),
In well #2 using FOXM1-A01-9-663 (SEQ ID NO: 53) (e),
In well #5 (f) using FOXM1-A01-9-494 (SEQ ID NO: 55),
In well #2 (g) using FOXM1-A01-9-341 (SEQ ID NO: 20),
In well #1 (h) using FOXM1-A01-10-566 (SEQ ID NO: 56),
In well #2 using FOXM1-A01-10-263 (SEQ ID NO: 57) (i),
In well #4 (j) using FOXM1-A01-10-308 (SEQ ID NO: 58),
In well #6 (k) using FOXM1-A01-10-232 (SEQ ID NO: 59),
In well #6 (l) and using FOXM1-A01-10-663 (SEQ ID NO: 60)
In well #6 (m) using FOXM1-A01-10-341 (SEQ ID NO: 61).
Meanwhile, specific IFN-gamma production was not observed for the other peptides shown in tables 1a and 1 b. For example, NO specific IFN-gamma production (n) was observed for FOXM1-A01-10-265 (SEQ ID NO: 42). As a result, although all peptides had the potential to bind HLA-A 01:01, 13 peptides were selected as peptides having CTL-inducing ability.
Establishment of CTL lines and clones specific for HLA-A 01:01 restricted FOXM1 derived peptides
CTL lines were established by expanding cells in well 1# and showed specific IFN-gamma production in the IFN-gamma ELISPOT assay for FOXM1-A01-10-566 (SEQ ID NO: 56). IFN-gamma production was observed for CTL lines against target cells (C1R-A01) impacted with FOXM1-A01-10-566 (SEQ ID NO: 56) as a result of measurement of IFN-gamma by ELISA. Furthermore, CTL clones were established by limiting dilution method. CTL clones stimulated with FOXM1-A01-9-233 (SEQ ID NO: 48) (a) or FOXM1-A01-10-566 (SEQ ID NO: 56) (b) each showed peptide-specific IFN-gamma production as a result of measurement of IFN-gamma by ELISA (FIG. 7).
IFN-gamma production against target cells expressing FOXM1 and HLA-A 01:01
IFN-gamma production was studied for specific CTL clones of FOXM1-A01-10-566 (SEQ ID NO: 56) against target cells expressing FOXM1 and HLA-A 01:01. COS7 cells expressing both FOXM1 and HLA-A 01:01 were prepared as target cells. COS7 cells expressing one of FOXM1 and HLA-A 01:01 were prepared as negative control cells. FOXM1-A01-10-566 (SEQ ID NO: 56) specific CTL clones showed IFN-gamma production against COS7 cells expressing both FOXM1 and HLA-A 01:01 (FIG. 8). On the other hand, no significant IFN-gamma production was observed against the negative control cells. This clearly demonstrates that FOXM1-a01-10-566 (SEQ ID NO: 56) is a peptide produced by antigen processing and is presented on the cell surface with HLA-A 01:01 molecules and recognized by CTLs. The results indicate that FOXM1-a01-10-566 (SEQ ID NO: 56) can be useful as a cancer vaccine for patients with enhanced FOXM1 expression in cancer cells.
Homology analysis of antigenic peptides
FOXM1-A01-9-233(SEQ ID NO:48)、FOXM1-A01-9-539(SEQ ID NO:49)、FOXM1-A01-9-631(SEQ ID NO:50)、FOXM1-A01-9-231(SEQ ID NO:52)、FOXM1-A01-9-663(SEQ ID NO:53)、FOXM1-A01-9-494(SEQ ID NO:55)、FOXM1-A01-9-341(SEQ ID NO:20)、FOXM1-A01-10-566(SEQ ID NO:56)、FOXM1-A01-10-263(SEQ ID NO:57)、FOXM1-A01-10-308(SEQ ID NO:58)、FOXM1-A01-10-232(SEQ ID NO:59)、FOXM1-A01-10-663(SEQ ID NO:60) And FOXM1-A01-10-341 (SEQ ID NO: 61) have been shown to induce CTLs exhibiting peptide-specific IFN-gamma production. Thus, to confirm that FOXM1-A01-9-233(SEQ ID NO:48)、FOXM1-A01-9-539(SEQ ID NO:49)、FOXM1-A01-9-631(SEQ ID NO:50)、FOXM1-A01-9-231(SEQ ID NO:52)、FOXM1-A01-9-663(SEQ ID NO:53)、FOXM1-A01-9-494(SEQ ID NO:55)、FOXM1-A01-9-341(SEQ ID NO:20)、FOXM1-A01-10-566(SEQ ID NO:56)、FOXM1-A01-10-263(SEQ ID NO:57)、FOXM1-A01-10-308(SEQ ID NO:58)、FOXM1-A01-10-232(SEQ ID NO:59)、FOXM1-A01-10-663(SEQ ID NO:60) and FOXM1-A01-10-341 (SEQ ID NO: 61) sequences were derived from FOXM1 only, homology analysis of peptide sequences was performed using the BLAST algorithm (BLAST. Ncbi. Nlm. Nih. Gov/BLAST. Cgi). As a result, FOXM1-A01-9-233(SEQ ID NO:48)、FOXM1-A01-9-539(SEQ ID NO:49)、FOXM1-A01-9-631(SEQ ID NO:50)、FOXM1-A01-9-231(SEQ ID NO:52)、FOXM1-A01-9-663(SEQ ID NO:53)、FOXM1-A01-9-494(SEQ ID NO:55)、FOXM1-A01-9-341(SEQ ID NO:20)、FOXM1-A01-10-566(SEQ ID NO:56)、FOXM1-A01-10-263(SEQ ID NO:57)、FOXM1-A01-10-308(SEQ ID NO:58)、FOXM1-A01-10-232(SEQ ID NO:59)、FOXM1-A01-10-663(SEQ ID NO:60) and FOXM1-A01-10-341 (SEQ ID NO: 61) were found only in FOXM 1. Thus, to the knowledge of the present inventors, these peptides are specific for FOXM1, making it almost impossible for these peptides to elicit an undesired immune response against molecules other than FOXM1, which are known to sensitize the human immune system. In summary, novel FOXM 1-derived HLA-A 01:01 restriction epitope peptides were identified. FOXM 1-derived epitope peptides have been demonstrated to be suitable for cancer immunotherapy.
Example 3
Preparation of emulsion formulations
The peptide was dissolved in a solvent for injection or sterile physiological saline to 1.0mg/ml to 10.0mg/ml, and collected into a syringe. This is connected via a connector to a syringe filled with IFA in an amount equivalent to that of the injected solvent or sterile physiological saline, and then mixed by alternately pushing the syringe plungers of the two connected syringes. After mixing for a few minutes, the completion of the emulsion was evaluated by drop test method (drop test method). The drip test method can be performed by dripping a drop of the mixed sample onto water. The emulsion was evaluated as complete when the sample dropped on water did not immediately spread in water, and the emulsion was evaluated as incomplete when the sample dropped on water immediately spread in water. When the emulsion is evaluated as incomplete, further mixing is performed to complete the emulsion. The finished emulsion may be administered to a cancer patient by subcutaneous injection. The cancer patient undergoing administration may be selected from patients affected by Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, testicular tumor, and the like.
Preparation of freeze-dried formulations
The peptide was dissolved in a solvent for injection to 1.0mg/ml to 10.0mg/ml, and filter sterilized. It was filled into sterilized vials and semi-closed with sterile rubber stoppers. After freeze-drying the vial, it was completely closed and seamed with an aluminum cap to produce a freeze-dried formulation. When used, an injection solvent or sterile physiological saline is injected into the vial to redissolve the freeze-dried powder. The redissolved solution in the vial was collected using a syringe, and the syringe was connected via a connector to a syringe filled with the same amount of IFA as the amount of redissolved solution collected. The redissolved solution and IFA were mixed by alternately pushing the syringe plungers of the two connected syringes. After mixing for a few minutes, the completion of the emulsion was evaluated by drop test method (drop test method). The finished emulsion may be administered to a cancer patient by subcutaneous injection. The cancer patient undergoing administration may be selected from patients affected by Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, testicular tumor, and the like.
Industrial applicability
The present invention provides novel FOXM 1-derived HLA-A 33-and HLA-A 01-restricted epitope peptides that induce strong and specific anti-tumor immune responses and thus have wide applicability to cancer types. The peptides, compositions, APCs and CTLs of the present invention are useful as peptide vaccines against FOXM 1-expressing cancers, such as Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, and testicular tumor.
While the invention has been described herein in detail and with respect to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. One skilled in the art will readily recognize from such routine experimentation that various changes and modifications can be made to the invention without departing from the spirit and scope of the invention, the scope and spirit of which is defined by the appended claims.
The invention includes
1. A peptide of less than 15 amino acids having cytotoxic T Cell (CTL) inducibility comprising an amino acid sequence selected from the group consisting of:
(a) Amino acid sequences selected from the group consisting of 1,2, 3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45, 46, 48, 49, 50, 52, 53, 55, 56, 57, 58, 59, 60 and 61, and
(B) Amino acid sequences ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61 in which 1, 2 or several amino acids are substituted, deleted, inserted and/or added to the amino acid sequences selected from the following groups.
2. The peptide of item 1, selected from the following groups (i) to (ii):
(i) A peptide comprising an amino acid sequence comprising one or more substitutions selected from the group consisting of SEQ ID NOs 1,2,3, 6, 7, 11, 12, 17, 18, 20, 22, 24, 26, 32, 33, 36, 39, 41, 42, 45 and 46 introduced into an amino acid sequence selected from the group consisting of:
(a) Substitution of the first amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid;
(b) Substitution of the second amino acid from the N-terminus with an amino acid selected from the group consisting of phenylalanine, tyrosine, alanine, isoleucine, leucine and valine, and
(C) Substitution of the C-terminal amino acid with an amino acid selected from the group consisting of arginine and lysine;
(ii) A peptide comprising an amino acid sequence, wherein one or more substitutions selected from the group consisting of (a) to (c) below are introduced into the amino acid sequence selected from the group consisting of SEQ ID NOs:48, 49, 50, 52, 53, 55, 20, 56, 57, 58, 59, 60 and 61:
(a) Replacing a second amino acid from the N-terminus with an amino acid selected from the group consisting of threonine and serine;
(b) Substitution of the third amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid, and
(C) The C-terminal amino acid was replaced with tyrosine.
3. The peptide of item 1, consisting of an amino acid sequence selected from the group consisting of ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61.
4. A polynucleotide encoding the peptide of any one of claims 1 to 3.
5. A composition comprising a pharmaceutically acceptable carrier and at least one ingredient selected from the following groups (a) to (e):
(a) One or more types of the peptide of any one of items 1 to 3;
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of items 1 to 3;
(c) An Antigen Presenting Cell (APC) that presents on its cell surface a complex of the peptide of any one of claims 1 to 3 and an HLA antigen;
(d) Exosomes presenting on their cell surfaces complexes of the peptides of any one of items 1 to 3 with HLA antigens, and
(E) A CTL targeting the peptide of any one of claims 1 to 3.
6. The composition of item 5, which is a composition for inducing CTLs, wherein the ingredient is at least one ingredient selected from the following groups (a) to (d):
(a) One or more types of the peptide of any one of items 1 to 3;
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of items 1 to 3;
(c) An Antigen Presenting Cell (APC) which presents a complex of the peptide of any one of items 1 to 3 and an HLA antigen on its cell surface, and
(D) Exosomes presenting on their cell surfaces complexes of the peptide of any one of claims 1 to 3 with HLA antigens.
7. The composition of item 5, which is a pharmaceutical composition.
8. The composition of item 7 for use in one or more of the uses selected from the group consisting of (i) cancer treatment, (ii) cancer prevention (prophltaxis) and (iii) prevention (prevention) of postoperative cancer recurrence.
9. The composition of item 7 for inducing an immune response against cancer.
10. The composition of item 8 or 9, wherein the cancer is selected from the group consisting of Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, diffuse gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, and testicular tumor.
11. The composition of any one of items 5 to 10 formulated for administration to at least one HLA-positive subject selected from the group consisting of HLA-a33 and HLA-a01.
12. A method of inducing APC having CTL inducibility comprising the step of:
(a) Contacting an APC with a peptide of any one of items 1 to 3 in vitro, ex vivo or in vivo, and
(B) Introducing a polynucleotide encoding the peptide of any one of claims 1 to 3 into an APC.
13. A method of inducing CTLs comprising the steps selected from the group consisting of:
(a) Co-culturing CD8 positive T cells with APCs that present on their surface a complex of HLA antigens and the peptide of any one of items 1 to 3;
(b) Co-culturing a CD 8-positive T cell with exosomes presenting complexes of HLA antigens with the peptide of any one of items 1 to 3 on their surface, and
(C) Introducing a polynucleotide encoding each subunit of a T Cell Receptor (TCR) capable of binding to a peptide of any one of items 1 to 3 presented on the cell surface by HLA antigens into a CD8 positive T cell.
An apc which presents on its surface a complex of an HLA antigen and a peptide of any of items 1 to 3.
15. The APC of item 14, which is induced by the method of item 12.
Ctl targeting the peptide of any one of claims 1-3.
17. CTL of item 16, which is induced by the method of item 13.
18. A method of inducing an immune response against cancer comprising administering to a subject at least one component selected from the group consisting of (a) to (e):
(a) One or more types of the peptide of any one of items 1 to 3;
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of items 1 to 3;
(c) An APC which presents on its cell surface a complex of the peptide of any one of claims 1 to 3 and an HLA antigen;
(d) Exosomes presenting on their cell surfaces complexes of the peptides of any one of items 1 to 3 with HLA antigens, and
(E) A CTL targeting the peptide of any one of claims 1 to 3.
19. A method of treating and/or preventing cancer, and/or preventing postoperative recurrence thereof, the method comprising administering to a subject at least one ingredient selected from the group consisting of (a) to (e):
(a) One or more types of the peptide of any one of items 1 to 3;
(b) One or more types of polynucleotides encoding in expressible form the peptide of any one of items 1 to 3;
(c) An APC which presents on its cell surface a complex of the peptide of any one of claims 1 to 3 and an HLA antigen;
(d) Exosomes presenting on their cell surfaces complexes of the peptides of any one of items 1 to 3 with HLA antigens, and
(E) A CTL targeting the peptide of any one of claims 1 to 3.
20. An antibody that binds to the peptide of any one of claims 1 to 3.
21. A method of screening for peptides having CTL inducibility, comprising the steps of:
(a) Generating a candidate sequence consisting of an amino acid sequence, wherein one, two or several amino acid residues ：SEQ ID NOs:1、2、3、6、7、11、12、17、18、20、22、24、26、32、33、36、39、41、42、45、46、48、49、50、52、53、55、56、57、58、59、60 and 61 are substituted, deleted, inserted and/or added to the original amino acid sequence consisting of an amino acid sequence selected from the group consisting of;
(b) Selecting a candidate sequence having no significant homology (sequence identity) with any known human gene product other than FOXM1 from the candidate sequences generated in (a);
(c) Contacting the APC with a peptide consisting of the candidate sequence selected in (b);
(d) Contacting the APC of (c) with a CD 8-positive T cell, and
(E) Peptides having equal or higher CTL inducibility than peptides consisting of the original amino acid sequence were selected.
22. An emulsion comprising one or more types of the peptide of any one of items 1 to 3, a water-soluble carrier and an oil adjuvant.
23. A kit comprising a container containing the composition of any one of items 5 to 11 and a container containing an adjuvant.
Sequence listing
<110> Tumor therapy science Co., ltd
<120> FOXM 1-derived peptides and vaccines containing the same
<130> ONC-A1604P
<150> JP 2015-200221
<151> 2015-10-08
<160> 76
<170> PatentIn version 3.5
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<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 17
Tyr Ser Gly Leu Arg Ser Pro Thr Arg
1 5
<210> 18
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 18
Asp Gln Val Phe Lys Gln Gln Lys Arg
1 5
<210> 19
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 19
Ser Glu Leu Ala Arg His Ser Lys Arg
1 5
<210> 20
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 20
Lys Thr Glu Leu Pro Leu Gly Ala Arg
1 5
<210> 21
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 21
Phe Ser Glu Gly Pro Ser Thr Ser Arg
1 5
<210> 22
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 22
Glu Met Leu Val Ile Gln His Arg Glu Arg
1 5 10
<210> 23
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 23
Glu Thr Leu Gly Pro Lys Pro Ala Ala Arg
1 5 10
<210> 24
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 24
Asn Ser Leu Ser Asn Ile Gln Trp Leu Arg
1 5 10
<210> 25
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 25
Asn Leu Ser Leu His Asp Met Phe Val Arg
1 5 10
<210> 26
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 26
Ser Tyr Ser Gly Leu Arg Ser Pro Thr Arg
1 5 10
<210> 27
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 27
Met Leu Val Ile Gln His Arg Glu Arg Arg
1 5 10
<210> 28
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 28
Phe Trp Thr Ile His Pro Ser Ala Asn Arg
1 5 10
<210> 29
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 29
His Ser Lys Arg Val Arg Ile Ala Pro Lys
1 5 10
<210> 30
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 30
Ile Gln Phe Ala Ile Asn Ser Thr Glu Arg
1 5 10
<210> 31
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 31
His Phe Pro Tyr Phe Lys His Ile Ala Lys
1 5 10
<210> 32
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 32
Gln Thr Gln Thr Ser Tyr Asp Ala Lys Arg
1 5 10
<210> 33
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 33
Ser Thr Pro Ser Lys Ser Val Leu Pro Arg
1 5 10
<210> 34
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 34
Glu Leu Ala Arg His Ser Lys Arg Val Arg
1 5 10
<210> 35
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 35
Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg
1 5 10
<210> 36
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 36
Leu Phe Ser Glu Gly Pro Ser Thr Ser Arg
1 5 10
<210> 37
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 37
Ser Ser Glu Leu Ala Arg His Ser Lys Arg
1 5 10
<210> 38
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 38
Ala Ser Trp Gln Asn Ser Val Ser Glu Arg
1 5 10
<210> 39
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 39
Met Ser Ser Glu Leu Ala Arg His Ser Lys
1 5 10
<210> 40
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 40
Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg
1 5 10
<210> 41
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 41
Glu Ser Trp Arg Leu Thr Pro Pro Ala Lys
1 5 10
<210> 42
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 42
Trp Ile Glu Asp His Phe Pro Tyr Phe Lys
1 5 10
<210> 43
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 43
Arg Pro Lys Lys Ser Tyr Ser Gly Leu Arg
1 5 10
<210> 44
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 44
Glu Trp Pro Ser Pro Ala Pro Ser Phe Lys
1 5 10
<210> 45
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 45
Ser Pro Arg Arg Pro Leu Ile Leu Lys Arg
1 5 10
<210> 46
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 46
Ala Ser Leu Met Ser Ser Glu Leu Ala Arg
1 5 10
<210> 47
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 47
Ser Ala Pro Pro Leu Glu Ser Pro Gln Arg
1 5 10
<210> 48
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 48
Val Ser Glu Arg Pro Pro Tyr Ser Tyr
1 5
<210> 49
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 49
Cys Val Asp Glu Pro Glu Leu Leu Phe
1 5
<210> 50
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 50
Ala Ser Asp Pro Leu Pro Asp Pro Leu
1 5
<210> 51
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 51
Leu Thr Glu Gly Leu Val Leu Asp Thr
1 5
<210> 52
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 52
Asn Ser Val Ser Glu Arg Pro Pro Tyr
1 5
<210> 53
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 53
Ser Ser Glu Pro Leu Asp Leu Ile Ser
1 5
<210> 54
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 54
Ser Ser Glu Leu Ala Arg His Ser Lys
1 5
<210> 55
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 55
Pro Thr Pro Arg Pro Lys Lys Ser Tyr
1 5
<210> 56
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 56
Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr
1 5 10
<210> 57
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 57
Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr
1 5 10
<210> 58
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 58
Trp Thr Ile His Pro Ser Ala Asn Arg Tyr
1 5 10
<210> 59
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 59
Ser Val Ser Glu Arg Pro Pro Tyr Ser Tyr
1 5 10
<210> 60
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 60
Ser Ser Glu Pro Leu Asp Leu Ile Ser Val
1 5 10
<210> 61
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Peptides derived from FOXM1
<400> 61
Lys Thr Glu Leu Pro Leu Gly Ala Arg Arg
1 5 10
<210> 62
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> PCR primers for TCR analysis
<400> 62
gtctaccagg cattcgcttc at 22
<210> 63
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> PCR primers for TCR analysis
<400> 63
tcagctggac cacagccgca gcgt 24
<210> 64
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> PCR primers for TCR analysis
<400> 64
tcagaaatcc tttctcttga c 21
<210> 65
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> PCR primers for TCR analysis
<400> 65
ctagcctctg gaatcctttc tctt 24
<210> 66
<211> 3506
<212> DNA
<213> Chile person
<220>
<221> CDS
<222> (284)..(2530)
<400> 66
tttcaaacag cggaacaaac tgaaagctcc ggtgccagac cccacccccg gccccggccc 60
gggaccccct cccctcccgg gatcccccgg ggttcccacc ccgcccgcac cgccggggac 120
ccggccggtc cggcgcgagc ccccgtccgg ggccctggct cggcccccag gttggaggag 180
cccggagccc gccttcggag ctacggccta acggcggcgg cgactgcagt ctggagggtc 240
cacacttgtg attctcaatg gagagtgaaa acgcagattc ata atg aaa act agc 295
Met Lys Thr Ser
1
ccc cgt cgg cca ctg att ctc aaa aga cgg agg ctg ccc ctt cct gtt 343
Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu Pro Leu Pro Val
5 10 15 20
caa aat gcc cca agt gaa aca tca gag gag gaa cct aag aga tcc cct 391
Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro Lys Arg Ser Pro
25 30 35
gcc caa cag gag tct aat caa gca gag gcc tcc aag gaa gtg gca gag 439
Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys Glu Val Ala Glu
40 45 50
tcc aac tct tgc aag ttt cca gct ggg atc aag att att aac cac ccc 487
Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile Ile Asn His Pro
55 60 65
acc atg ccc aac acg caa gta gtg gcc atc ccc aac aat gct aat att 535
Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn Asn Ala Asn Ile
70 75 80
cac agc atc atc aca gca ctg act gcc aag gga aaa gag agt ggc agt 583
His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys Glu Ser Gly Ser
85 90 95 100
agt ggg ccc aac aaa ttc atc ctc atc agc tgt ggg gga gcc cca act 631
Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly Gly Ala Pro Thr
105 110 115
cag cct cca gga ctc cgg cct caa acc caa acc agc tat gat gcc aaa 679
Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser Tyr Asp Ala Lys
120 125 130
agg aca gaa gtg acc ctg gag acc ttg gga cca aaa cct gca gct agg 727
Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys Pro Ala Ala Arg
135 140 145
gat gtg aat ctt cct aga cca cct gga gcc ctt tgc gag cag aaa cgg 775
Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys Glu Gln Lys Arg
150 155 160
gag acc tgt gca gat ggt gag gca gca ggc tgc act atc aac aat agc 823
Glu Thr Cys Ala Asp Gly Glu Ala Ala Gly Cys Thr Ile Asn Asn Ser
165 170 175 180
cta tcc aac atc cag tgg ctt cga aag atg agt tct gat gga ctg ggc 871
Leu Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser Asp Gly Leu Gly
185 190 195
tcc cgc agc atc aag caa gag atg gag gaa aag gag aat tgt cac ctg 919
Ser Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu Asn Cys His Leu
200 205 210
gag cag cga cag gtt aag gtt gag gag cct tcg aga cca tca gcg tcc 967
Glu Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg Pro Ser Ala Ser
215 220 225
tgg cag aac tct gtg tct gag cgg cca ccc tac tct tac atg gcc atg 1015
Trp Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser Tyr Met Ala Met
230 235 240
ata caa ttc gcc atc aac agc act gag agg aag cgc atg act ttg aaa 1063
Ile Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg Met Thr Leu Lys
245 250 255 260
gac atc tat acg tgg att gag gac cac ttt ccc tac ttt aag cac att 1111
Asp Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr Phe Lys His Ile
265 270 275
gcc aag cca ggc tgg aag aac tcc atc cgc cac aac ctt tcc ctg cac 1159
Ala Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn Leu Ser Leu His
280 285 290
gac atg ttt gtc cgg gag acg tct gcc aat ggc aag gtc tcc ttc tgg 1207
Asp Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys Val Ser Phe Trp
295 300 305
acc att cac ccc agt gcc aac cgc tac ttg aca ttg gac cag gtg ttt 1255
Thr Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu Asp Gln Val Phe
310 315 320
aag cag cag aaa cga ccg aat cca gag ctc cgc cgg aac atg acc atc 1303
Lys Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg Asn Met Thr Ile
325 330 335 340
aaa acc gaa ctc ccc ctg ggc gca cgg cgg aag atg aag cca ctg cta 1351
Lys Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met Lys Pro Leu Leu
345 350 355
cca cgg gtc agc tca tac ctg gta cct atc cag ttc ccg gtg aac cag 1399
Pro Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe Pro Val Asn Gln
360 365 370
tca ctg gtg ttg cag ccc tcg gtg aag gtg cca ttg ccc ctg gcg gct 1447
Ser Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu Pro Leu Ala Ala
375 380 385
tcc ctc atg agc tca gag ctt gcc cgc cat agc aag cga gtc cgc att 1495
Ser Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys Arg Val Arg Ile
390 395 400
gcc ccc aag gtg ctg cta gct gag gag ggg ata gct cct ctt tct tct 1543
Ala Pro Lys Val Leu Leu Ala Glu Glu Gly Ile Ala Pro Leu Ser Ser
405 410 415 420
gca gga cca ggg aaa gag gag aaa ctc ctg ttt gga gaa ggg ttt tct 1591
Ala Gly Pro Gly Lys Glu Glu Lys Leu Leu Phe Gly Glu Gly Phe Ser
425 430 435
cct ttg ctt cca gtt cag act atc aag gag gaa gaa atc cag cct ggg 1639
Pro Leu Leu Pro Val Gln Thr Ile Lys Glu Glu Glu Ile Gln Pro Gly
440 445 450
gag gaa atg cca cac tta gcg aga ccc atc aaa gtg gag agc cct ccc 1687
Glu Glu Met Pro His Leu Ala Arg Pro Ile Lys Val Glu Ser Pro Pro
455 460 465
ttg gaa gag tgg ccc tcc ccg gcc cca tct ttc aaa gag gaa tca tct 1735
Leu Glu Glu Trp Pro Ser Pro Ala Pro Ser Phe Lys Glu Glu Ser Ser
470 475 480
cac tcc tgg gag gat tcg tcc caa tct ccc acc cca aga ccc aag aag 1783
His Ser Trp Glu Asp Ser Ser Gln Ser Pro Thr Pro Arg Pro Lys Lys
485 490 495 500
tcc tac agt ggg ctt agg tcc cca acc cgg tgt gtc tcg gaa atg ctt 1831
Ser Tyr Ser Gly Leu Arg Ser Pro Thr Arg Cys Val Ser Glu Met Leu
505 510 515
gtg att caa cac agg gag agg agg gag agg agc cgg tct cgg agg aaa 1879
Val Ile Gln His Arg Glu Arg Arg Glu Arg Ser Arg Ser Arg Arg Lys
520 525 530
cag cat cta ctg cct ccc tgt gtg gat gag ccg gag ctg ctc ttc tca 1927
Gln His Leu Leu Pro Pro Cys Val Asp Glu Pro Glu Leu Leu Phe Ser
535 540 545
gag ggg ccc agt act tcc cgc tgg gcc gca gag ctc ccg ttc cca gca 1975
Glu Gly Pro Ser Thr Ser Arg Trp Ala Ala Glu Leu Pro Phe Pro Ala
550 555 560
gac tcc tct gac cct gcc tcc cag ctc agc tac tcc cag gaa gtg gga 2023
Asp Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr Ser Gln Glu Val Gly
565 570 575 580
gga cct ttt aag aca ccc att aag gaa acg ctg ccc atc tcc tcc acc 2071
Gly Pro Phe Lys Thr Pro Ile Lys Glu Thr Leu Pro Ile Ser Ser Thr
585 590 595
ccg agc aaa tct gtc ctc ccc aga acc cct gaa tcc tgg agg ctc acg 2119
Pro Ser Lys Ser Val Leu Pro Arg Thr Pro Glu Ser Trp Arg Leu Thr
600 605 610
ccc cca gcc aaa gta ggg gga ctg gat ttc agc cca gta caa acc tcc 2167
Pro Pro Ala Lys Val Gly Gly Leu Asp Phe Ser Pro Val Gln Thr Ser
615 620 625
cag ggt gcc tct gac ccc ttg cct gac ccc ctg ggg ctg atg gat ctc 2215
Gln Gly Ala Ser Asp Pro Leu Pro Asp Pro Leu Gly Leu Met Asp Leu
630 635 640
agc acc act ccc ttg caa agt gct ccc ccc ctt gaa tca ccg caa agg 2263
Ser Thr Thr Pro Leu Gln Ser Ala Pro Pro Leu Glu Ser Pro Gln Arg
645 650 655 660
ctc ctc agt tca gaa ccc tta gac ctc atc tcc gtc ccc ttt ggc aac 2311
Leu Leu Ser Ser Glu Pro Leu Asp Leu Ile Ser Val Pro Phe Gly Asn
665 670 675
tct tct ccc tca gat ata gac gtc ccc aag cca ggc tcc ccg gag cca 2359
Ser Ser Pro Ser Asp Ile Asp Val Pro Lys Pro Gly Ser Pro Glu Pro
680 685 690
cag gtt tct ggc ctt gca gcc aat cgt tct ctg aca gaa ggc ctg gtc 2407
Gln Val Ser Gly Leu Ala Ala Asn Arg Ser Leu Thr Glu Gly Leu Val
695 700 705
ctg gac aca atg aat gac agc ctc agc aag atc ctg ctg gac atc agc 2455
Leu Asp Thr Met Asn Asp Ser Leu Ser Lys Ile Leu Leu Asp Ile Ser
710 715 720
ttt cct ggc ctg gac gag gac cca ctg ggc cct gac aac atc aac tgg 2503
Phe Pro Gly Leu Asp Glu Asp Pro Leu Gly Pro Asp Asn Ile Asn Trp
725 730 735 740
tcc cag ttt att cct gag cta cag tag agccctgccc ttgcccctgt 2550
Ser Gln Phe Ile Pro Glu Leu Gln
745
gctcaagctg tccaccatcc cgggcactcc aaggctcagt gcaccccaag cctctgagtg 2610
aggacagcag gcagggactg ttctgctcct catagctccc tgctgcctga ttatgcaaaa 2670
gtagcagtca caccctagcc actgctggga ccttgtgttc cccaagagta tctgattcct 2730
ctgctgtccc tgccaggagc tgaagggtgg gaacaacaaa ggcaatggtg aaaagagatt 2790
aggaaccccc cagcctgttt ccattctctg cccagcagtc tcttaccttc cctgatcttt 2850
gcagggtggt ccgtgtaaat agtataaatt ctccaaatta tcctctaatt ataaatgtaa 2910
gcttatttcc ttagatcatt atccagagac tgccagaagg tgggtaggat gacctggggt 2970
ttcaattgac ttctgttcct tgcttttagt tttgatagaa gggaagacct gcagtgcacg 3030
gtttcttcca ggctgaggta cctggatctt gggttcttca ctgcagggac ccagacaagt 3090
ggatctgctt gccagagtcc tttttgcccc tccctgccac ctccccgtgt ttccaagtca 3150
gctttcctgc aagaagaaat cctggttaaa aaagtctttt gtattgggtc aggagttgaa 3210
tttggggtgg gaggatggat gcaactgaag cagagtgtgg gtgcccagat gtgcgctatt 3270
agatgtttct ctgataatgt ccccaatcat accagggaga ctggcattga cgagaactca 3330
ggtggaggct tgagaaggcc gaaagggccc ctgacctgcc tggcttcctt agcttgcccc 3390
tcagctttgc aaagagccac cctaggcccc agctgaccgc atgggtgtga gccagcttga 3450
gaacactaac tactcaataa aagcgaaggt ggacatgaaa aaaaaaaaaa aaaaaa 3506
<210> 67
<211> 748
<212> PRT
<213> Chile person
<400> 67
Met Lys Thr Ser Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu
1 5 10 15
Pro Leu Pro Val Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro
20 25 30
Lys Arg Ser Pro Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys
35 40 45
Glu Val Ala Glu Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile
50 55 60
Ile Asn His Pro Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn
65 70 75 80
Asn Ala Asn Ile His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys
85 90 95
Glu Ser Gly Ser Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly
100 105 110
Gly Ala Pro Thr Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser
115 120 125
Tyr Asp Ala Lys Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys
130 135 140
Pro Ala Ala Arg Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys
145 150 155 160
Glu Gln Lys Arg Glu Thr Cys Ala Asp Gly Glu Ala Ala Gly Cys Thr
165 170 175
Ile Asn Asn Ser Leu Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser
180 185 190
Asp Gly Leu Gly Ser Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu
195 200 205
Asn Cys His Leu Glu Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg
210 215 220
Pro Ser Ala Ser Trp Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser
225 230 235 240
Tyr Met Ala Met Ile Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg
245 250 255
Met Thr Leu Lys Asp Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr
260 265 270
Phe Lys His Ile Ala Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn
275 280 285
Leu Ser Leu His Asp Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys
290 295 300
Val Ser Phe Trp Thr Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu
305 310 315 320
Asp Gln Val Phe Lys Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg
325 330 335
Asn Met Thr Ile Lys Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met
340 345 350
Lys Pro Leu Leu Pro Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe
355 360 365
Pro Val Asn Gln Ser Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu
370 375 380
Pro Leu Ala Ala Ser Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys
385 390 395 400
Arg Val Arg Ile Ala Pro Lys Val Leu Leu Ala Glu Glu Gly Ile Ala
405 410 415
Pro Leu Ser Ser Ala Gly Pro Gly Lys Glu Glu Lys Leu Leu Phe Gly
420 425 430
Glu Gly Phe Ser Pro Leu Leu Pro Val Gln Thr Ile Lys Glu Glu Glu
435 440 445
Ile Gln Pro Gly Glu Glu Met Pro His Leu Ala Arg Pro Ile Lys Val
450 455 460
Glu Ser Pro Pro Leu Glu Glu Trp Pro Ser Pro Ala Pro Ser Phe Lys
465 470 475 480
Glu Glu Ser Ser His Ser Trp Glu Asp Ser Ser Gln Ser Pro Thr Pro
485 490 495
Arg Pro Lys Lys Ser Tyr Ser Gly Leu Arg Ser Pro Thr Arg Cys Val
500 505 510
Ser Glu Met Leu Val Ile Gln His Arg Glu Arg Arg Glu Arg Ser Arg
515 520 525
Ser Arg Arg Lys Gln His Leu Leu Pro Pro Cys Val Asp Glu Pro Glu
530 535 540
Leu Leu Phe Ser Glu Gly Pro Ser Thr Ser Arg Trp Ala Ala Glu Leu
545 550 555 560
Pro Phe Pro Ala Asp Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr Ser
565 570 575
Gln Glu Val Gly Gly Pro Phe Lys Thr Pro Ile Lys Glu Thr Leu Pro
580 585 590
Ile Ser Ser Thr Pro Ser Lys Ser Val Leu Pro Arg Thr Pro Glu Ser
595 600 605
Trp Arg Leu Thr Pro Pro Ala Lys Val Gly Gly Leu Asp Phe Ser Pro
610 615 620
Val Gln Thr Ser Gln Gly Ala Ser Asp Pro Leu Pro Asp Pro Leu Gly
625 630 635 640
Leu Met Asp Leu Ser Thr Thr Pro Leu Gln Ser Ala Pro Pro Leu Glu
645 650 655
Ser Pro Gln Arg Leu Leu Ser Ser Glu Pro Leu Asp Leu Ile Ser Val
660 665 670
Pro Phe Gly Asn Ser Ser Pro Ser Asp Ile Asp Val Pro Lys Pro Gly
675 680 685
Ser Pro Glu Pro Gln Val Ser Gly Leu Ala Ala Asn Arg Ser Leu Thr
690 695 700
Glu Gly Leu Val Leu Asp Thr Met Asn Asp Ser Leu Ser Lys Ile Leu
705 710 715 720
Leu Asp Ile Ser Phe Pro Gly Leu Asp Glu Asp Pro Leu Gly Pro Asp
725 730 735
Asn Ile Asn Trp Ser Gln Phe Ile Pro Glu Leu Gln
740 745
<210> 68
<211> 3506
<212> DNA
<213> Chile person
<220>
<221> CDS
<222> (284)..(2530)
<400> 68
tttcaaacag cggaacaaac tgaaagctcc ggtgccagac cccacccccg gccccggccc 60
gggaccccct cccctcccgg gatcccccgg ggttcccacc ccgcccgcac cgccggggac 120
ccggccggtc cggcgcgagc ccccgtccgg ggccctggct cggcccccag gttggaggag 180
cccggagccc gccttcggag ctacggccta acggcggcgg cgactgcagt ctggagggtc 240
cacacttgtg attctcaatg gagagtgaaa acgcagattc ata atg aaa act agc 295
Met Lys Thr Ser
1
ccc cgt cgg cca ctg att ctc aaa aga cgg agg ctg ccc ctt cct gtt 343
Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu Pro Leu Pro Val
5 10 15 20
caa aat gcc cca agt gaa aca tca gag gag gaa cct aag aga tcc cct 391
Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro Lys Arg Ser Pro
25 30 35
gcc caa cag gag tct aat caa gca gag gcc tcc aag gaa gtg gca gag 439
Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys Glu Val Ala Glu
40 45 50
tcc aac tct tgc aag ttt cca gct ggg atc aag att att aac cac ccc 487
Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile Ile Asn His Pro
55 60 65
acc atg ccc aac acg caa gta gtg gcc atc ccc aac aat gct aat att 535
Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn Asn Ala Asn Ile
70 75 80
cac agc atc atc aca gca ctg act gcc aag gga aaa gag agt ggc agt 583
His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys Glu Ser Gly Ser
85 90 95 100
agt ggg ccc aac aaa ttc atc ctc atc agc tgt ggg gga gcc cca act 631
Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly Gly Ala Pro Thr
105 110 115
cag cct cca gga ctc cgg cct caa acc caa acc agc tat gat gcc aaa 679
Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser Tyr Asp Ala Lys
120 125 130
agg aca gaa gtg acc ctg gag acc ttg gga cca aaa cct gca gct agg 727
Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys Pro Ala Ala Arg
135 140 145
gat gtg aat ctt cct aga cca cct gga gcc ctt tgc gag cag aaa cgg 775
Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys Glu Gln Lys Arg
150 155 160
gag acc tgt gat ggt gag gca gca ggc tgc act atc aac aat agc cta 823
Glu Thr Cys Asp Gly Glu Ala Ala Gly Cys Thr Ile Asn Asn Ser Leu
165 170 175 180
tcc aac atc cag tgg ctt cga aag atg agt tct gat gga ctg ggc tcc 871
Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser Asp Gly Leu Gly Ser
185 190 195
cgc agc atc aag caa gag atg gag gaa aag gag aat tgt cac ctg gag 919
Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu Asn Cys His Leu Glu
200 205 210
cag cga cag gtt aag gtt gag gag cct tcg aga cca tca gcg tcc tgg 967
Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg Pro Ser Ala Ser Trp
215 220 225
cag aac tct gtg tct gag cgg cca ccc tac tct tac atg gcc atg ata 1015
Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser Tyr Met Ala Met Ile
230 235 240
caa ttc gcc atc aac agc act gag agg aag cgc atg act ttg aaa gac 1063
Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg Met Thr Leu Lys Asp
245 250 255 260
atc tat acg tgg att gag gac cac ttt ccc tac ttt aag cac att gcc 1111
Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr Phe Lys His Ile Ala
265 270 275
aag cca ggc tgg aag aac tcc atc cgc cac aac ctt tcc ctg cac gac 1159
Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn Leu Ser Leu His Asp
280 285 290
atg ttt gtc cgg gag acg tct gcc aat ggc aag gtc tcc ttc tgg acc 1207
Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys Val Ser Phe Trp Thr
295 300 305
att cac ccc agt gcc aac cgc tac ttg aca ttg gac cag gtg ttt aag 1255
Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu Asp Gln Val Phe Lys
310 315 320
cag cag cag aaa cga ccg aat cca gag ctc cgc cgg aac atg acc atc 1303
Gln Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg Asn Met Thr Ile
325 330 335 340
aaa acc gaa ctc ccc ctg ggc gca cgg cgg aag atg aag cca ctg cta 1351
Lys Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met Lys Pro Leu Leu
345 350 355
cca cgg gtc agc tca tac ctg gta cct atc cag ttc ccg gtg aac cag 1399
Pro Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe Pro Val Asn Gln
360 365 370
tca ctg gtg ttg cag ccc tcg gtg aag gtg cca ttg ccc ctg gcg gct 1447
Ser Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu Pro Leu Ala Ala
375 380 385
tcc ctc atg agc tca gag ctt gcc cgc cat agc aag cga gtc cgc att 1495
Ser Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys Arg Val Arg Ile
390 395 400
gcc ccc aag gtg ctg cta gct gag gag ggg ata gct cct ctt tct tct 1543
Ala Pro Lys Val Leu Leu Ala Glu Glu Gly Ile Ala Pro Leu Ser Ser
405 410 415 420
gca gga cca ggg aaa gag gag aaa ctc ctg ttt gga gaa ggg ttt tct 1591
Ala Gly Pro Gly Lys Glu Glu Lys Leu Leu Phe Gly Glu Gly Phe Ser
425 430 435
cct ttg ctt cca gtt cag act atc aag gag gaa gaa atc cag cct ggg 1639
Pro Leu Leu Pro Val Gln Thr Ile Lys Glu Glu Glu Ile Gln Pro Gly
440 445 450
gag gaa atg cca cac tta gcg aga ccc atc aaa gtg gag agc cct ccc 1687
Glu Glu Met Pro His Leu Ala Arg Pro Ile Lys Val Glu Ser Pro Pro
455 460 465
ttg gaa gag tgg ccc tcc ccg gcc cca tct ttc aaa gag gaa tca tct 1735
Leu Glu Glu Trp Pro Ser Pro Ala Pro Ser Phe Lys Glu Glu Ser Ser
470 475 480
cac tcc tgg gag gat tcg tcc caa tct ccc acc cca aga ccc aag aag 1783
His Ser Trp Glu Asp Ser Ser Gln Ser Pro Thr Pro Arg Pro Lys Lys
485 490 495 500
tcc tac agt ggg ctt agg tcc cca acc cgg tgt gtc tcg gaa atg ctt 1831
Ser Tyr Ser Gly Leu Arg Ser Pro Thr Arg Cys Val Ser Glu Met Leu
505 510 515
gtg att caa cac agg gag agg agg gag agg agc cgg tct cgg agg aaa 1879
Val Ile Gln His Arg Glu Arg Arg Glu Arg Ser Arg Ser Arg Arg Lys
520 525 530
cag cat cta ctg cct ccc tgt gtg gat gag ccg gag ctg ctc ttc tca 1927
Gln His Leu Leu Pro Pro Cys Val Asp Glu Pro Glu Leu Leu Phe Ser
535 540 545
gag ggg ccc agt act tcc cgc tgg gcc gca gag ctc ccg ttc cca gca 1975
Glu Gly Pro Ser Thr Ser Arg Trp Ala Ala Glu Leu Pro Phe Pro Ala
550 555 560
gac tcc tct gac cct gcc tcc cag ctc agc tac tcc cag gaa gtg gga 2023
Asp Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr Ser Gln Glu Val Gly
565 570 575 580
gga cct ttt aag aca ccc att aag gaa acg ctg ccc atc tcc tcc acc 2071
Gly Pro Phe Lys Thr Pro Ile Lys Glu Thr Leu Pro Ile Ser Ser Thr
585 590 595
ccg agc aaa tct gtc ctc ccc aga acc cct gaa tcc tgg agg ctc acg 2119
Pro Ser Lys Ser Val Leu Pro Arg Thr Pro Glu Ser Trp Arg Leu Thr
600 605 610
ccc cca gcc aaa gta ggg gga ctg gat ttc agc cca gta caa acc tcc 2167
Pro Pro Ala Lys Val Gly Gly Leu Asp Phe Ser Pro Val Gln Thr Ser
615 620 625
cag ggt gcc tct gac ccc ttg cct gac ccc ctg ggg ctg atg gat ctc 2215
Gln Gly Ala Ser Asp Pro Leu Pro Asp Pro Leu Gly Leu Met Asp Leu
630 635 640
agc acc act ccc ttg caa agt gct ccc ccc ctt gaa tca ccg caa agg 2263
Ser Thr Thr Pro Leu Gln Ser Ala Pro Pro Leu Glu Ser Pro Gln Arg
645 650 655 660
ctc ctc agt tca gaa ccc tta gac ctc atc tcc gtc ccc ttt ggc aac 2311
Leu Leu Ser Ser Glu Pro Leu Asp Leu Ile Ser Val Pro Phe Gly Asn
665 670 675
tct tct ccc tca gat ata gac gtc ccc aag cca ggc tcc ccg gag cca 2359
Ser Ser Pro Ser Asp Ile Asp Val Pro Lys Pro Gly Ser Pro Glu Pro
680 685 690
cag gtt tct ggc ctt gca gcc aat cgt tct ctg aca gaa ggc ctg gtc 2407
Gln Val Ser Gly Leu Ala Ala Asn Arg Ser Leu Thr Glu Gly Leu Val
695 700 705
ctg gac aca atg aat gac agc ctc agc aag atc ctg ctg gac atc agc 2455
Leu Asp Thr Met Asn Asp Ser Leu Ser Lys Ile Leu Leu Asp Ile Ser
710 715 720
ttt cct ggc ctg gac gag gac cca ctg ggc cct gac aac atc aac tgg 2503
Phe Pro Gly Leu Asp Glu Asp Pro Leu Gly Pro Asp Asn Ile Asn Trp
725 730 735 740
tcc cag ttt att cct gag cta cag tag agccctgccc ttgcccctgt 2550
Ser Gln Phe Ile Pro Glu Leu Gln
745
gctcaagctg tccaccatcc cgggcactcc aaggctcagt gcaccccaag cctctgagtg 2610
aggacagcag gcagggactg ttctgctcct catagctccc tgctgcctga ttatgcaaaa 2670
gtagcagtca caccctagcc actgctggga ccttgtgttc cccaagagta tctgattcct 2730
ctgctgtccc tgccaggagc tgaagggtgg gaacaacaaa ggcaatggtg aaaagagatt 2790
aggaaccccc cagcctgttt ccattctctg cccagcagtc tcttaccttc cctgatcttt 2850
gcagggtggt ccgtgtaaat agtataaatt ctccaaatta tcctctaatt ataaatgtaa 2910
gcttatttcc ttagatcatt atccagagac tgccagaagg tgggtaggat gacctggggt 2970
ttcaattgac ttctgttcct tgcttttagt tttgatagaa gggaagacct gcagtgcacg 3030
gtttcttcca ggctgaggta cctggatctt gggttcttca ctgcagggac ccagacaagt 3090
ggatctgctt gccagagtcc tttttgcccc tccctgccac ctccccgtgt ttccaagtca 3150
gctttcctgc aagaagaaat cctggttaaa aaagtctttt gtattgggtc aggagttgaa 3210
tttggggtgg gaggatggat gcaactgaag cagagtgtgg gtgcccagat gtgcgctatt 3270
agatgtttct ctgataatgt ccccaatcat accagggaga ctggcattga cgagaactca 3330
ggtggaggct tgagaaggcc gaaagggccc ctgacctgcc tggcttcctt agcttgcccc 3390
tcagctttgc aaagagccac cctaggcccc agctgaccgc atgggtgtga gccagcttga 3450
gaacactaac tactcaataa aagcgaaggt ggacatgaaa aaaaaaaaaa aaaaaa 3506
<210> 69
<211> 748
<212> PRT
<213> Chile person
<400> 69
Met Lys Thr Ser Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu
1 5 10 15
Pro Leu Pro Val Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro
20 25 30
Lys Arg Ser Pro Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys
35 40 45
Glu Val Ala Glu Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile
50 55 60
Ile Asn His Pro Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn
65 70 75 80
Asn Ala Asn Ile His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys
85 90 95
Glu Ser Gly Ser Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly
100 105 110
Gly Ala Pro Thr Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser
115 120 125
Tyr Asp Ala Lys Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys
130 135 140
Pro Ala Ala Arg Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys
145 150 155 160
Glu Gln Lys Arg Glu Thr Cys Asp Gly Glu Ala Ala Gly Cys Thr Ile
165 170 175
Asn Asn Ser Leu Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser Asp
180 185 190
Gly Leu Gly Ser Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu Asn
195 200 205
Cys His Leu Glu Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg Pro
210 215 220
Ser Ala Ser Trp Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser Tyr
225 230 235 240
Met Ala Met Ile Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg Met
245 250 255
Thr Leu Lys Asp Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr Phe
260 265 270
Lys His Ile Ala Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn Leu
275 280 285
Ser Leu His Asp Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys Val
290 295 300
Ser Phe Trp Thr Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu Asp
305 310 315 320
Gln Val Phe Lys Gln Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg
325 330 335
Asn Met Thr Ile Lys Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met
340 345 350
Lys Pro Leu Leu Pro Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe
355 360 365
Pro Val Asn Gln Ser Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu
370 375 380
Pro Leu Ala Ala Ser Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys
385 390 395 400
Arg Val Arg Ile Ala Pro Lys Val Leu Leu Ala Glu Glu Gly Ile Ala
405 410 415
Pro Leu Ser Ser Ala Gly Pro Gly Lys Glu Glu Lys Leu Leu Phe Gly
420 425 430
Glu Gly Phe Ser Pro Leu Leu Pro Val Gln Thr Ile Lys Glu Glu Glu
435 440 445
Ile Gln Pro Gly Glu Glu Met Pro His Leu Ala Arg Pro Ile Lys Val
450 455 460
Glu Ser Pro Pro Leu Glu Glu Trp Pro Ser Pro Ala Pro Ser Phe Lys
465 470 475 480
Glu Glu Ser Ser His Ser Trp Glu Asp Ser Ser Gln Ser Pro Thr Pro
485 490 495
Arg Pro Lys Lys Ser Tyr Ser Gly Leu Arg Ser Pro Thr Arg Cys Val
500 505 510
Ser Glu Met Leu Val Ile Gln His Arg Glu Arg Arg Glu Arg Ser Arg
515 520 525
Ser Arg Arg Lys Gln His Leu Leu Pro Pro Cys Val Asp Glu Pro Glu
530 535 540
Leu Leu Phe Ser Glu Gly Pro Ser Thr Ser Arg Trp Ala Ala Glu Leu
545 550 555 560
Pro Phe Pro Ala Asp Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr Ser
565 570 575
Gln Glu Val Gly Gly Pro Phe Lys Thr Pro Ile Lys Glu Thr Leu Pro
580 585 590
Ile Ser Ser Thr Pro Ser Lys Ser Val Leu Pro Arg Thr Pro Glu Ser
595 600 605
Trp Arg Leu Thr Pro Pro Ala Lys Val Gly Gly Leu Asp Phe Ser Pro
610 615 620
Val Gln Thr Ser Gln Gly Ala Ser Asp Pro Leu Pro Asp Pro Leu Gly
625 630 635 640
Leu Met Asp Leu Ser Thr Thr Pro Leu Gln Ser Ala Pro Pro Leu Glu
645 650 655
Ser Pro Gln Arg Leu Leu Ser Ser Glu Pro Leu Asp Leu Ile Ser Val
660 665 670
Pro Phe Gly Asn Ser Ser Pro Ser Asp Ile Asp Val Pro Lys Pro Gly
675 680 685
Ser Pro Glu Pro Gln Val Ser Gly Leu Ala Ala Asn Arg Ser Leu Thr
690 695 700
Glu Gly Leu Val Leu Asp Thr Met Asn Asp Ser Leu Ser Lys Ile Leu
705 710 715 720
Leu Asp Ile Ser Phe Pro Gly Leu Asp Glu Asp Pro Leu Gly Pro Asp
725 730 735
Asn Ile Asn Trp Ser Gln Phe Ile Pro Glu Leu Gln
740 745
<210> 70
<211> 3503
<212> DNA
<213> Chile person
<220>
<221> CDS
<222> (284)..(2527)
<400> 70
tttcaaacag cggaacaaac tgaaagctcc ggtgccagac cccacccccg gccccggccc 60
gggaccccct cccctcccgg gatcccccgg ggttcccacc ccgcccgcac cgccggggac 120
ccggccggtc cggcgcgagc ccccgtccgg ggccctggct cggcccccag gttggaggag 180
cccggagccc gccttcggag ctacggccta acggcggcgg cgactgcagt ctggagggtc 240
cacacttgtg attctcaatg gagagtgaaa acgcagattc ata atg aaa act agc 295
Met Lys Thr Ser
1
ccc cgt cgg cca ctg att ctc aaa aga cgg agg ctg ccc ctt cct gtt 343
Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu Pro Leu Pro Val
5 10 15 20
caa aat gcc cca agt gaa aca tca gag gag gaa cct aag aga tcc cct 391
Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro Lys Arg Ser Pro
25 30 35
gcc caa cag gag tct aat caa gca gag gcc tcc aag gaa gtg gca gag 439
Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys Glu Val Ala Glu
40 45 50
tcc aac tct tgc aag ttt cca gct ggg atc aag att att aac cac ccc 487
Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile Ile Asn His Pro
55 60 65
acc atg ccc aac acg caa gta gtg gcc atc ccc aac aat gct aat att 535
Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn Asn Ala Asn Ile
70 75 80
cac agc atc atc aca gca ctg act gcc aag gga aaa gag agt ggc agt 583
His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys Glu Ser Gly Ser
85 90 95 100
agt ggg ccc aac aaa ttc atc ctc atc agc tgt ggg gga gcc cca act 631
Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly Gly Ala Pro Thr
105 110 115
cag cct cca gga ctc cgg cct caa acc caa acc agc tat gat gcc aaa 679
Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser Tyr Asp Ala Lys
120 125 130
agg aca gaa gtg acc ctg gag acc ttg gga cca aaa cct gca gct agg 727
Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys Pro Ala Ala Arg
135 140 145
gat gtg aat ctt cct aga cca cct gga gcc ctt tgc gag cag aaa cgg 775
Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys Glu Gln Lys Arg
150 155 160
gag acc tgt gat ggt gag gca gca ggc tgc act atc aac aat agc cta 823
Glu Thr Cys Asp Gly Glu Ala Ala Gly Cys Thr Ile Asn Asn Ser Leu
165 170 175 180
tcc aac atc cag tgg ctt cga aag atg agt tct gat gga ctg ggc tcc 871
Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser Asp Gly Leu Gly Ser
185 190 195
cgc agc atc aag caa gag atg gag gaa aag gag aat tgt cac ctg gag 919
Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu Asn Cys His Leu Glu
200 205 210
cag cga cag gtt aag gtt gag gag cct tcg aga cca tca gcg tcc tgg 967
Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg Pro Ser Ala Ser Trp
215 220 225
cag aac tct gtg tct gag cgg cca ccc tac tct tac atg gcc atg ata 1015
Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser Tyr Met Ala Met Ile
230 235 240
caa ttc gcc atc aac agc act gag agg aag cgc atg act ttg aaa gac 1063
Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg Met Thr Leu Lys Asp
245 250 255 260
atc tat acg tgg att gag gac cac ttt ccc tac ttt aag cac att gcc 1111
Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr Phe Lys His Ile Ala
265 270 275
aag cca ggc tgg aag aac tcc atc cgc cac aac ctt tcc ctg cac gac 1159
Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn Leu Ser Leu His Asp
280 285 290
atg ttt gtc cgg gag acg tct gcc aat ggc aag gtc tcc ttc tgg acc 1207
Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys Val Ser Phe Trp Thr
295 300 305
att cac ccc agt gcc aac cgc tac ttg aca ttg gac cag gtg ttt aag 1255
Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu Asp Gln Val Phe Lys
310 315 320
cag cag aaa cga ccg aat cca gag ctc cgc cgg aac atg acc atc aaa 1303
Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg Asn Met Thr Ile Lys
325 330 335 340
acc gaa ctc ccc ctg ggc gca cgg cgg aag atg aag cca ctg cta cca 1351
Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met Lys Pro Leu Leu Pro
345 350 355
cgg gtc agc tca tac ctg gta cct atc cag ttc ccg gtg aac cag tca 1399
Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe Pro Val Asn Gln Ser
360 365 370
ctg gtg ttg cag ccc tcg gtg aag gtg cca ttg ccc ctg gcg gct tcc 1447
Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu Pro Leu Ala Ala Ser
375 380 385
ctc atg agc tca gag ctt gcc cgc cat agc aag cga gtc cgc att gcc 1495
Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys Arg Val Arg Ile Ala
390 395 400
ccc aag gtg ctg cta gct gag gag ggg ata gct cct ctt tct tct gca 1543
Pro Lys Val Leu Leu Ala Glu Glu Gly Ile Ala Pro Leu Ser Ser Ala
405 410 415 420
gga cca ggg aaa gag gag aaa ctc ctg ttt gga gaa ggg ttt tct cct 1591
Gly Pro Gly Lys Glu Glu Lys Leu Leu Phe Gly Glu Gly Phe Ser Pro
425 430 435
ttg ctt cca gtt cag act atc aag gag gaa gaa atc cag cct ggg gag 1639
Leu Leu Pro Val Gln Thr Ile Lys Glu Glu Glu Ile Gln Pro Gly Glu
440 445 450
gaa atg cca cac tta gcg aga ccc atc aaa gtg gag agc cct ccc ttg 1687
Glu Met Pro His Leu Ala Arg Pro Ile Lys Val Glu Ser Pro Pro Leu
455 460 465
gaa gag tgg ccc tcc ccg gcc cca tct ttc aaa gag gaa tca tct cac 1735
Glu Glu Trp Pro Ser Pro Ala Pro Ser Phe Lys Glu Glu Ser Ser His
470 475 480
tcc tgg gag gat tcg tcc caa tct ccc acc cca aga ccc aag aag tcc 1783
Ser Trp Glu Asp Ser Ser Gln Ser Pro Thr Pro Arg Pro Lys Lys Ser
485 490 495 500
tac agt ggg ctt agg tcc cca acc cgg tgt gtc tcg gaa atg ctt gtg 1831
Tyr Ser Gly Leu Arg Ser Pro Thr Arg Cys Val Ser Glu Met Leu Val
505 510 515
att caa cac agg gag agg agg gag agg agc cgg tct cgg agg aaa cag 1879
Ile Gln His Arg Glu Arg Arg Glu Arg Ser Arg Ser Arg Arg Lys Gln
520 525 530
cat cta ctg cct ccc tgt gtg gat gag ccg gag ctg ctc ttc tca gag 1927
His Leu Leu Pro Pro Cys Val Asp Glu Pro Glu Leu Leu Phe Ser Glu
535 540 545
ggg ccc agt act tcc cgc tgg gcc gca gag ctc ccg ttc cca gca gac 1975
Gly Pro Ser Thr Ser Arg Trp Ala Ala Glu Leu Pro Phe Pro Ala Asp
550 555 560
tcc tct gac cct gcc tcc cag ctc agc tac tcc cag gaa gtg gga gga 2023
Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr Ser Gln Glu Val Gly Gly
565 570 575 580
cct ttt aag aca ccc att aag gaa acg ctg ccc atc tcc tcc acc ccg 2071
Pro Phe Lys Thr Pro Ile Lys Glu Thr Leu Pro Ile Ser Ser Thr Pro
585 590 595
agc aaa tct gtc ctc ccc aga acc cct gaa tcc tgg agg ctc acg ccc 2119
Ser Lys Ser Val Leu Pro Arg Thr Pro Glu Ser Trp Arg Leu Thr Pro
600 605 610
cca gcc aaa gta ggg gga ctg gat ttc agc cca gta caa acc tcc cag 2167
Pro Ala Lys Val Gly Gly Leu Asp Phe Ser Pro Val Gln Thr Ser Gln
615 620 625
ggt gcc tct gac ccc ttg cct gac ccc ctg ggg ctg atg gat ctc agc 2215
Gly Ala Ser Asp Pro Leu Pro Asp Pro Leu Gly Leu Met Asp Leu Ser
630 635 640
acc act ccc ttg caa agt gct ccc ccc ctt gaa tca ccg caa agg ctc 2263
Thr Thr Pro Leu Gln Ser Ala Pro Pro Leu Glu Ser Pro Gln Arg Leu
645 650 655 660
ctc agt tca gaa ccc tta gac ctc atc tcc gtc ccc ttt ggc aac tct 2311
Leu Ser Ser Glu Pro Leu Asp Leu Ile Ser Val Pro Phe Gly Asn Ser
665 670 675
tct ccc tca gat ata gac gtc ccc aag cca ggc tcc ccg gag cca cag 2359
Ser Pro Ser Asp Ile Asp Val Pro Lys Pro Gly Ser Pro Glu Pro Gln
680 685 690
gtt tct ggc ctt gca gcc aat cgt tct ctg aca gaa ggc ctg gtc ctg 2407
Val Ser Gly Leu Ala Ala Asn Arg Ser Leu Thr Glu Gly Leu Val Leu
695 700 705
gac aca atg aat gac agc ctc agc aag atc ctg ctg gac atc agc ttt 2455
Asp Thr Met Asn Asp Ser Leu Ser Lys Ile Leu Leu Asp Ile Ser Phe
710 715 720
cct ggc ctg gac gag gac cca ctg ggc cct gac aac atc aac tgg tcc 2503
Pro Gly Leu Asp Glu Asp Pro Leu Gly Pro Asp Asn Ile Asn Trp Ser
725 730 735 740
cag ttt att cct gag cta cag tag agccctgccc ttgcccctgt gctcaagctg 2557
Gln Phe Ile Pro Glu Leu Gln
745
tccaccatcc cgggcactcc aaggctcagt gcaccccaag cctctgagtg aggacagcag 2617
gcagggactg ttctgctcct catagctccc tgctgcctga ttatgcaaaa gtagcagtca 2677
caccctagcc actgctggga ccttgtgttc cccaagagta tctgattcct ctgctgtccc 2737
tgccaggagc tgaagggtgg gaacaacaaa ggcaatggtg aaaagagatt aggaaccccc 2797
cagcctgttt ccattctctg cccagcagtc tcttaccttc cctgatcttt gcagggtggt 2857
ccgtgtaaat agtataaatt ctccaaatta tcctctaatt ataaatgtaa gcttatttcc 2917
ttagatcatt atccagagac tgccagaagg tgggtaggat gacctggggt ttcaattgac 2977
ttctgttcct tgcttttagt tttgatagaa gggaagacct gcagtgcacg gtttcttcca 3037
ggctgaggta cctggatctt gggttcttca ctgcagggac ccagacaagt ggatctgctt 3097
gccagagtcc tttttgcccc tccctgccac ctccccgtgt ttccaagtca gctttcctgc 3157
aagaagaaat cctggttaaa aaagtctttt gtattgggtc aggagttgaa tttggggtgg 3217
gaggatggat gcaactgaag cagagtgtgg gtgcccagat gtgcgctatt agatgtttct 3277
ctgataatgt ccccaatcat accagggaga ctggcattga cgagaactca ggtggaggct 3337
tgagaaggcc gaaagggccc ctgacctgcc tggcttcctt agcttgcccc tcagctttgc 3397
aaagagccac cctaggcccc agctgaccgc atgggtgtga gccagcttga gaacactaac 3457
tactcaataa aagcgaaggt ggacatgaaa aaaaaaaaaa aaaaaa 3503
<210> 71
<211> 747
<212> PRT
<213> Chile person
<400> 71
Met Lys Thr Ser Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu
1 5 10 15
Pro Leu Pro Val Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro
20 25 30
Lys Arg Ser Pro Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys
35 40 45
Glu Val Ala Glu Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile
50 55 60
Ile Asn His Pro Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn
65 70 75 80
Asn Ala Asn Ile His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys
85 90 95
Glu Ser Gly Ser Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly
100 105 110
Gly Ala Pro Thr Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser
115 120 125
Tyr Asp Ala Lys Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys
130 135 140
Pro Ala Ala Arg Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys
145 150 155 160
Glu Gln Lys Arg Glu Thr Cys Asp Gly Glu Ala Ala Gly Cys Thr Ile
165 170 175
Asn Asn Ser Leu Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser Asp
180 185 190
Gly Leu Gly Ser Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu Asn
195 200 205
Cys His Leu Glu Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg Pro
210 215 220
Ser Ala Ser Trp Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser Tyr
225 230 235 240
Met Ala Met Ile Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg Met
245 250 255
Thr Leu Lys Asp Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr Phe
260 265 270
Lys His Ile Ala Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn Leu
275 280 285
Ser Leu His Asp Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys Val
290 295 300
Ser Phe Trp Thr Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu Asp
305 310 315 320
Gln Val Phe Lys Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg Asn
325 330 335
Met Thr Ile Lys Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met Lys
340 345 350
Pro Leu Leu Pro Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe Pro
355 360 365
Val Asn Gln Ser Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu Pro
370 375 380
Leu Ala Ala Ser Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys Arg
385 390 395 400
Val Arg Ile Ala Pro Lys Val Leu Leu Ala Glu Glu Gly Ile Ala Pro
405 410 415
Leu Ser Ser Ala Gly Pro Gly Lys Glu Glu Lys Leu Leu Phe Gly Glu
420 425 430
Gly Phe Ser Pro Leu Leu Pro Val Gln Thr Ile Lys Glu Glu Glu Ile
435 440 445
Gln Pro Gly Glu Glu Met Pro His Leu Ala Arg Pro Ile Lys Val Glu
450 455 460
Ser Pro Pro Leu Glu Glu Trp Pro Ser Pro Ala Pro Ser Phe Lys Glu
465 470 475 480
Glu Ser Ser His Ser Trp Glu Asp Ser Ser Gln Ser Pro Thr Pro Arg
485 490 495
Pro Lys Lys Ser Tyr Ser Gly Leu Arg Ser Pro Thr Arg Cys Val Ser
500 505 510
Glu Met Leu Val Ile Gln His Arg Glu Arg Arg Glu Arg Ser Arg Ser
515 520 525
Arg Arg Lys Gln His Leu Leu Pro Pro Cys Val Asp Glu Pro Glu Leu
530 535 540
Leu Phe Ser Glu Gly Pro Ser Thr Ser Arg Trp Ala Ala Glu Leu Pro
545 550 555 560
Phe Pro Ala Asp Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr Ser Gln
565 570 575
Glu Val Gly Gly Pro Phe Lys Thr Pro Ile Lys Glu Thr Leu Pro Ile
580 585 590
Ser Ser Thr Pro Ser Lys Ser Val Leu Pro Arg Thr Pro Glu Ser Trp
595 600 605
Arg Leu Thr Pro Pro Ala Lys Val Gly Gly Leu Asp Phe Ser Pro Val
610 615 620
Gln Thr Ser Gln Gly Ala Ser Asp Pro Leu Pro Asp Pro Leu Gly Leu
625 630 635 640
Met Asp Leu Ser Thr Thr Pro Leu Gln Ser Ala Pro Pro Leu Glu Ser
645 650 655
Pro Gln Arg Leu Leu Ser Ser Glu Pro Leu Asp Leu Ile Ser Val Pro
660 665 670
Phe Gly Asn Ser Ser Pro Ser Asp Ile Asp Val Pro Lys Pro Gly Ser
675 680 685
Pro Glu Pro Gln Val Ser Gly Leu Ala Ala Asn Arg Ser Leu Thr Glu
690 695 700
Gly Leu Val Leu Asp Thr Met Asn Asp Ser Leu Ser Lys Ile Leu Leu
705 710 715 720
Asp Ile Ser Phe Pro Gly Leu Asp Glu Asp Pro Leu Gly Pro Asp Asn
725 730 735
Ile Asn Trp Ser Gln Phe Ile Pro Glu Leu Gln
740 745
<210> 72
<211> 3551
<212> DNA
<213> Chile person
<220>
<221> CDS
<222> (284)..(2575)
<400> 72
tttcaaacag cggaacaaac tgaaagctcc ggtgccagac cccacccccg gccccggccc 60
gggaccccct cccctcccgg gatcccccgg ggttcccacc ccgcccgcac cgccggggac 120
ccggccggtc cggcgcgagc ccccgtccgg ggccctggct cggcccccag gttggaggag 180
cccggagccc gccttcggag ctacggccta acggcggcgg cgactgcagt ctggagggtc 240
cacacttgtg attctcaatg gagagtgaaa acgcagattc ata atg aaa act agc 295
Met Lys Thr Ser
1
ccc cgt cgg cca ctg att ctc aaa aga cgg agg ctg ccc ctt cct gtt 343
Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu Pro Leu Pro Val
5 10 15 20
caa aat gcc cca agt gaa aca tca gag gag gaa cct aag aga tcc cct 391
Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro Lys Arg Ser Pro
25 30 35
gcc caa cag gag tct aat caa gca gag gcc tcc aag gaa gtg gca gag 439
Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys Glu Val Ala Glu
40 45 50
tcc aac tct tgc aag ttt cca gct ggg atc aag att att aac cac ccc 487
Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile Ile Asn His Pro
55 60 65
acc atg ccc aac acg caa gta gtg gcc atc ccc aac aat gct aat att 535
Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn Asn Ala Asn Ile
70 75 80
cac agc atc atc aca gca ctg act gcc aag gga aaa gag agt ggc agt 583
His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys Glu Ser Gly Ser
85 90 95 100
agt ggg ccc aac aaa ttc atc ctc atc agc tgt ggg gga gcc cca act 631
Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly Gly Ala Pro Thr
105 110 115
cag cct cca gga ctc cgg cct caa acc caa acc agc tat gat gcc aaa 679
Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser Tyr Asp Ala Lys
120 125 130
agg aca gaa gtg acc ctg gag acc ttg gga cca aaa cct gca gct agg 727
Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys Pro Ala Ala Arg
135 140 145
gat gtg aat ctt cct aga cca cct gga gcc ctt tgc gag cag aaa cgg 775
Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys Glu Gln Lys Arg
150 155 160
gag acc tgt gca gat ggt gag gca gca ggc tgc act atc aac aat agc 823
Glu Thr Cys Ala Asp Gly Glu Ala Ala Gly Cys Thr Ile Asn Asn Ser
165 170 175 180
cta tcc aac atc cag tgg ctt cga aag atg agt tct gat gga ctg ggc 871
Leu Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser Asp Gly Leu Gly
185 190 195
tcc cgc agc atc aag caa gag atg gag gaa aag gag aat tgt cac ctg 919
Ser Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu Asn Cys His Leu
200 205 210
gag cag cga cag gtt aag gtt gag gag cct tcg aga cca tca gcg tcc 967
Glu Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg Pro Ser Ala Ser
215 220 225
tgg cag aac tct gtg tct gag cgg cca ccc tac tct tac atg gcc atg 1015
Trp Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser Tyr Met Ala Met
230 235 240
ata caa ttc gcc atc aac agc act gag agg aag cgc atg act ttg aaa 1063
Ile Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg Met Thr Leu Lys
245 250 255 260
gac atc tat acg tgg att gag gac cac ttt ccc tac ttt aag cac att 1111
Asp Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr Phe Lys His Ile
265 270 275
gcc aag cca ggc tgg aag aac tcc atc cgc cac aac ctt tcc ctg cac 1159
Ala Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn Leu Ser Leu His
280 285 290
gac atg ttt gtc cgg gag acg tct gcc aat ggc aag gtc tcc ttc tgg 1207
Asp Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys Val Ser Phe Trp
295 300 305
acc att cac ccc agt gcc aac cgc tac ttg aca ttg gac cag gtg ttt 1255
Thr Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu Asp Gln Val Phe
310 315 320
aag cca ctg gac cca ggg tct cca caa ttg ccc gag cac ttg gaa tca 1303
Lys Pro Leu Asp Pro Gly Ser Pro Gln Leu Pro Glu His Leu Glu Ser
325 330 335 340
cag cag aaa cga ccg aat cca gag ctc cgc cgg aac atg acc atc aaa 1351
Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg Asn Met Thr Ile Lys
345 350 355
acc gaa ctc ccc ctg ggc gca cgg cgg aag atg aag cca ctg cta cca 1399
Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met Lys Pro Leu Leu Pro
360 365 370
cgg gtc agc tca tac ctg gta cct atc cag ttc ccg gtg aac cag tca 1447
Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe Pro Val Asn Gln Ser
375 380 385
ctg gtg ttg cag ccc tcg gtg aag gtg cca ttg ccc ctg gcg gct tcc 1495
Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu Pro Leu Ala Ala Ser
390 395 400
ctc atg agc tca gag ctt gcc cgc cat agc aag cga gtc cgc att gcc 1543
Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys Arg Val Arg Ile Ala
405 410 415 420
ccc aag gtg ctg cta gct gag gag ggg ata gct cct ctt tct tct gca 1591
Pro Lys Val Leu Leu Ala Glu Glu Gly Ile Ala Pro Leu Ser Ser Ala
425 430 435
gga cca ggg aaa gag gag aaa ctc ctg ttt gga gaa ggg ttt tct cct 1639
Gly Pro Gly Lys Glu Glu Lys Leu Leu Phe Gly Glu Gly Phe Ser Pro
440 445 450
ttg ctt cca gtt cag act atc aag gag gaa gaa atc cag cct ggg gag 1687
Leu Leu Pro Val Gln Thr Ile Lys Glu Glu Glu Ile Gln Pro Gly Glu
455 460 465
gaa atg cca cac tta gcg aga ccc atc aaa gtg gag agc cct ccc ttg 1735
Glu Met Pro His Leu Ala Arg Pro Ile Lys Val Glu Ser Pro Pro Leu
470 475 480
gaa gag tgg ccc tcc ccg gcc cca tct ttc aaa gag gaa tca tct cac 1783
Glu Glu Trp Pro Ser Pro Ala Pro Ser Phe Lys Glu Glu Ser Ser His
485 490 495 500
tcc tgg gag gat tcg tcc caa tct ccc acc cca aga ccc aag aag tcc 1831
Ser Trp Glu Asp Ser Ser Gln Ser Pro Thr Pro Arg Pro Lys Lys Ser
505 510 515
tac agt ggg ctt agg tcc cca acc cgg tgt gtc tcg gaa atg ctt gtg 1879
Tyr Ser Gly Leu Arg Ser Pro Thr Arg Cys Val Ser Glu Met Leu Val
520 525 530
att caa cac agg gag agg agg gag agg agc cgg tct cgg agg aaa cag 1927
Ile Gln His Arg Glu Arg Arg Glu Arg Ser Arg Ser Arg Arg Lys Gln
535 540 545
cat cta ctg cct ccc tgt gtg gat gag ccg gag ctg ctc ttc tca gag 1975
His Leu Leu Pro Pro Cys Val Asp Glu Pro Glu Leu Leu Phe Ser Glu
550 555 560
ggg ccc agt act tcc cgc tgg gcc gca gag ctc ccg ttc cca gca gac 2023
Gly Pro Ser Thr Ser Arg Trp Ala Ala Glu Leu Pro Phe Pro Ala Asp
565 570 575 580
tcc tct gac cct gcc tcc cag ctc agc tac tcc cag gaa gtg gga gga 2071
Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr Ser Gln Glu Val Gly Gly
585 590 595
cct ttt aag aca ccc att aag gaa acg ctg ccc atc tcc tcc acc ccg 2119
Pro Phe Lys Thr Pro Ile Lys Glu Thr Leu Pro Ile Ser Ser Thr Pro
600 605 610
agc aaa tct gtc ctc ccc aga acc cct gaa tcc tgg agg ctc acg ccc 2167
Ser Lys Ser Val Leu Pro Arg Thr Pro Glu Ser Trp Arg Leu Thr Pro
615 620 625
cca gcc aaa gta ggg gga ctg gat ttc agc cca gta caa acc tcc cag 2215
Pro Ala Lys Val Gly Gly Leu Asp Phe Ser Pro Val Gln Thr Ser Gln
630 635 640
ggt gcc tct gac ccc ttg cct gac ccc ctg ggg ctg atg gat ctc agc 2263
Gly Ala Ser Asp Pro Leu Pro Asp Pro Leu Gly Leu Met Asp Leu Ser
645 650 655 660
acc act ccc ttg caa agt gct ccc ccc ctt gaa tca ccg caa agg ctc 2311
Thr Thr Pro Leu Gln Ser Ala Pro Pro Leu Glu Ser Pro Gln Arg Leu
665 670 675
ctc agt tca gaa ccc tta gac ctc atc tcc gtc ccc ttt ggc aac tct 2359
Leu Ser Ser Glu Pro Leu Asp Leu Ile Ser Val Pro Phe Gly Asn Ser
680 685 690
tct ccc tca gat ata gac gtc ccc aag cca ggc tcc ccg gag cca cag 2407
Ser Pro Ser Asp Ile Asp Val Pro Lys Pro Gly Ser Pro Glu Pro Gln
695 700 705
gtt tct ggc ctt gca gcc aat cgt tct ctg aca gaa ggc ctg gtc ctg 2455
Val Ser Gly Leu Ala Ala Asn Arg Ser Leu Thr Glu Gly Leu Val Leu
710 715 720
gac aca atg aat gac agc ctc agc aag atc ctg ctg gac atc agc ttt 2503
Asp Thr Met Asn Asp Ser Leu Ser Lys Ile Leu Leu Asp Ile Ser Phe
725 730 735 740
cct ggc ctg gac gag gac cca ctg ggc cct gac aac atc aac tgg tcc 2551
Pro Gly Leu Asp Glu Asp Pro Leu Gly Pro Asp Asn Ile Asn Trp Ser
745 750 755
cag ttt att cct gag cta cag tag agccctgccc ttgcccctgt gctcaagctg 2605
Gln Phe Ile Pro Glu Leu Gln
760
tccaccatcc cgggcactcc aaggctcagt gcaccccaag cctctgagtg aggacagcag 2665
gcagggactg ttctgctcct catagctccc tgctgcctga ttatgcaaaa gtagcagtca 2725
caccctagcc actgctggga ccttgtgttc cccaagagta tctgattcct ctgctgtccc 2785
tgccaggagc tgaagggtgg gaacaacaaa ggcaatggtg aaaagagatt aggaaccccc 2845
cagcctgttt ccattctctg cccagcagtc tcttaccttc cctgatcttt gcagggtggt 2905
ccgtgtaaat agtataaatt ctccaaatta tcctctaatt ataaatgtaa gcttatttcc 2965
ttagatcatt atccagagac tgccagaagg tgggtaggat gacctggggt ttcaattgac 3025
ttctgttcct tgcttttagt tttgatagaa gggaagacct gcagtgcacg gtttcttcca 3085
ggctgaggta cctggatctt gggttcttca ctgcagggac ccagacaagt ggatctgctt 3145
gccagagtcc tttttgcccc tccctgccac ctccccgtgt ttccaagtca gctttcctgc 3205
aagaagaaat cctggttaaa aaagtctttt gtattgggtc aggagttgaa tttggggtgg 3265
gaggatggat gcaactgaag cagagtgtgg gtgcccagat gtgcgctatt agatgtttct 3325
ctgataatgt ccccaatcat accagggaga ctggcattga cgagaactca ggtggaggct 3385
tgagaaggcc gaaagggccc ctgacctgcc tggcttcctt agcttgcccc tcagctttgc 3445
aaagagccac cctaggcccc agctgaccgc atgggtgtga gccagcttga gaacactaac 3505
tactcaataa aagcgaaggt ggacatgaaa aaaaaaaaaa aaaaaa 3551
<210> 73
<211> 763
<212> PRT
<213> Chile person
<400> 73
Met Lys Thr Ser Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu
1 5 10 15
Pro Leu Pro Val Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro
20 25 30
Lys Arg Ser Pro Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys
35 40 45
Glu Val Ala Glu Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile
50 55 60
Ile Asn His Pro Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn
65 70 75 80
Asn Ala Asn Ile His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys
85 90 95
Glu Ser Gly Ser Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly
100 105 110
Gly Ala Pro Thr Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser
115 120 125
Tyr Asp Ala Lys Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys
130 135 140
Pro Ala Ala Arg Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys
145 150 155 160
Glu Gln Lys Arg Glu Thr Cys Ala Asp Gly Glu Ala Ala Gly Cys Thr
165 170 175
Ile Asn Asn Ser Leu Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser
180 185 190
Asp Gly Leu Gly Ser Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu
195 200 205
Asn Cys His Leu Glu Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg
210 215 220
Pro Ser Ala Ser Trp Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser
225 230 235 240
Tyr Met Ala Met Ile Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg
245 250 255
Met Thr Leu Lys Asp Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr
260 265 270
Phe Lys His Ile Ala Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn
275 280 285
Leu Ser Leu His Asp Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys
290 295 300
Val Ser Phe Trp Thr Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu
305 310 315 320
Asp Gln Val Phe Lys Pro Leu Asp Pro Gly Ser Pro Gln Leu Pro Glu
325 330 335
His Leu Glu Ser Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg Asn
340 345 350
Met Thr Ile Lys Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met Lys
355 360 365
Pro Leu Leu Pro Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe Pro
370 375 380
Val Asn Gln Ser Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu Pro
385 390 395 400
Leu Ala Ala Ser Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys Arg
405 410 415
Val Arg Ile Ala Pro Lys Val Leu Leu Ala Glu Glu Gly Ile Ala Pro
420 425 430
Leu Ser Ser Ala Gly Pro Gly Lys Glu Glu Lys Leu Leu Phe Gly Glu
435 440 445
Gly Phe Ser Pro Leu Leu Pro Val Gln Thr Ile Lys Glu Glu Glu Ile
450 455 460
Gln Pro Gly Glu Glu Met Pro His Leu Ala Arg Pro Ile Lys Val Glu
465 470 475 480
Ser Pro Pro Leu Glu Glu Trp Pro Ser Pro Ala Pro Ser Phe Lys Glu
485 490 495
Glu Ser Ser His Ser Trp Glu Asp Ser Ser Gln Ser Pro Thr Pro Arg
500 505 510
Pro Lys Lys Ser Tyr Ser Gly Leu Arg Ser Pro Thr Arg Cys Val Ser
515 520 525
Glu Met Leu Val Ile Gln His Arg Glu Arg Arg Glu Arg Ser Arg Ser
530 535 540
Arg Arg Lys Gln His Leu Leu Pro Pro Cys Val Asp Glu Pro Glu Leu
545 550 555 560
Leu Phe Ser Glu Gly Pro Ser Thr Ser Arg Trp Ala Ala Glu Leu Pro
565 570 575
Phe Pro Ala Asp Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr Ser Gln
580 585 590
Glu Val Gly Gly Pro Phe Lys Thr Pro Ile Lys Glu Thr Leu Pro Ile
595 600 605
Ser Ser Thr Pro Ser Lys Ser Val Leu Pro Arg Thr Pro Glu Ser Trp
610 615 620
Arg Leu Thr Pro Pro Ala Lys Val Gly Gly Leu Asp Phe Ser Pro Val
625 630 635 640
Gln Thr Ser Gln Gly Ala Ser Asp Pro Leu Pro Asp Pro Leu Gly Leu
645 650 655
Met Asp Leu Ser Thr Thr Pro Leu Gln Ser Ala Pro Pro Leu Glu Ser
660 665 670
Pro Gln Arg Leu Leu Ser Ser Glu Pro Leu Asp Leu Ile Ser Val Pro
675 680 685
Phe Gly Asn Ser Ser Pro Ser Asp Ile Asp Val Pro Lys Pro Gly Ser
690 695 700
Pro Glu Pro Gln Val Ser Gly Leu Ala Ala Asn Arg Ser Leu Thr Glu
705 710 715 720
Gly Leu Val Leu Asp Thr Met Asn Asp Ser Leu Ser Lys Ile Leu Leu
725 730 735
Asp Ile Ser Phe Pro Gly Leu Asp Glu Asp Pro Leu Gly Pro Asp Asn
740 745 750
Ile Asn Trp Ser Gln Phe Ile Pro Glu Leu Gln
755 760
<210> 74
<211> 3665
<212> DNA
<213> Chile person
<220>
<221> CDS
<222> (284)..(2689)
<400> 74
tttcaaacag cggaacaaac tgaaagctcc ggtgccagac cccacccccg gccccggccc 60
gggaccccct cccctcccgg gatcccccgg ggttcccacc ccgcccgcac cgccggggac 120
ccggccggtc cggcgcgagc ccccgtccgg ggccctggct cggcccccag gttggaggag 180
cccggagccc gccttcggag ctacggccta acggcggcgg cgactgcagt ctggagggtc 240
cacacttgtg attctcaatg gagagtgaaa acgcagattc ata atg aaa act agc 295
Met Lys Thr Ser
1
ccc cgt cgg cca ctg att ctc aaa aga cgg agg ctg ccc ctt cct gtt 343
Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu Pro Leu Pro Val
5 10 15 20
caa aat gcc cca agt gaa aca tca gag gag gaa cct aag aga tcc cct 391
Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro Lys Arg Ser Pro
25 30 35
gcc caa cag gag tct aat caa gca gag gcc tcc aag gaa gtg gca gag 439
Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys Glu Val Ala Glu
40 45 50
tcc aac tct tgc aag ttt cca gct ggg atc aag att att aac cac ccc 487
Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile Ile Asn His Pro
55 60 65
acc atg ccc aac acg caa gta gtg gcc atc ccc aac aat gct aat att 535
Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn Asn Ala Asn Ile
70 75 80
cac agc atc atc aca gca ctg act gcc aag gga aaa gag agt ggc agt 583
His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys Glu Ser Gly Ser
85 90 95 100
agt ggg ccc aac aaa ttc atc ctc atc agc tgt ggg gga gcc cca act 631
Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly Gly Ala Pro Thr
105 110 115
cag cct cca gga ctc cgg cct caa acc caa acc agc tat gat gcc aaa 679
Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser Tyr Asp Ala Lys
120 125 130
agg aca gaa gtg acc ctg gag acc ttg gga cca aaa cct gca gct agg 727
Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys Pro Ala Ala Arg
135 140 145
gat gtg aat ctt cct aga cca cct gga gcc ctt tgc gag cag aaa cgg 775
Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys Glu Gln Lys Arg
150 155 160
gag acc tgt gca gat ggt gag gca gca ggc tgc act atc aac aat agc 823
Glu Thr Cys Ala Asp Gly Glu Ala Ala Gly Cys Thr Ile Asn Asn Ser
165 170 175 180
cta tcc aac atc cag tgg ctt cga aag atg agt tct gat gga ctg ggc 871
Leu Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser Asp Gly Leu Gly
185 190 195
tcc cgc agc atc aag caa gag atg gag gaa aag gag aat tgt cac ctg 919
Ser Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu Asn Cys His Leu
200 205 210
gag cag cga cag gtt aag gtt gag gag cct tcg aga cca tca gcg tcc 967
Glu Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg Pro Ser Ala Ser
215 220 225
tgg cag aac tct gtg tct gag cgg cca ccc tac tct tac atg gcc atg 1015
Trp Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser Tyr Met Ala Met
230 235 240
ata caa ttc gcc atc aac agc act gag agg aag cgc atg act ttg aaa 1063
Ile Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg Met Thr Leu Lys
245 250 255 260
gac atc tat acg tgg att gag gac cac ttt ccc tac ttt aag cac att 1111
Asp Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr Phe Lys His Ile
265 270 275
gcc aag cca ggc tgg aag aac tcc atc cgc cac aac ctt tcc ctg cac 1159
Ala Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn Leu Ser Leu His
280 285 290
gac atg ttt gtc cgg gag acg tct gcc aat ggc aag gtc tcc ttc tgg 1207
Asp Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys Val Ser Phe Trp
295 300 305
acc att cac ccc agt gcc aac cgc tac ttg aca ttg gac cag gtg ttt 1255
Thr Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu Asp Gln Val Phe
310 315 320
aag cca ctg gac cca ggg tct cca caa ttg ccc gag cac ttg gaa tca 1303
Lys Pro Leu Asp Pro Gly Ser Pro Gln Leu Pro Glu His Leu Glu Ser
325 330 335 340
cag cag aaa cga ccg aat cca gag ctc cgc cgg aac atg acc atc aaa 1351
Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg Asn Met Thr Ile Lys
345 350 355
acc gaa ctc ccc ctg ggc gca cgg cgg aag atg aag cca ctg cta cca 1399
Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met Lys Pro Leu Leu Pro
360 365 370
cgg gtc agc tca tac ctg gta cct atc cag ttc ccg gtg aac cag tca 1447
Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe Pro Val Asn Gln Ser
375 380 385
ctg gtg ttg cag ccc tcg gtg aag gtg cca ttg ccc ctg gcg gct tcc 1495
Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu Pro Leu Ala Ala Ser
390 395 400
ctc atg agc tca gag ctt gcc cgc cat agc aag cga gtc cgc att gcc 1543
Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys Arg Val Arg Ile Ala
405 410 415 420
ccc aag gtt ttt ggg gaa cag gtg gtg ttt ggt tac atg agt aag ttc 1591
Pro Lys Val Phe Gly Glu Gln Val Val Phe Gly Tyr Met Ser Lys Phe
425 430 435
ttt agt ggc gat ctg cga gat ttt ggt aca ccc atc acc agc ttg ttt 1639
Phe Ser Gly Asp Leu Arg Asp Phe Gly Thr Pro Ile Thr Ser Leu Phe
440 445 450
aat ttt atc ttt ctt tgt tta tca gtg ctg cta gct gag gag ggg ata 1687
Asn Phe Ile Phe Leu Cys Leu Ser Val Leu Leu Ala Glu Glu Gly Ile
455 460 465
gct cct ctt tct tct gca gga cca ggg aaa gag gag aaa ctc ctg ttt 1735
Ala Pro Leu Ser Ser Ala Gly Pro Gly Lys Glu Glu Lys Leu Leu Phe
470 475 480
gga gaa ggg ttt tct cct ttg ctt cca gtt cag act atc aag gag gaa 1783
Gly Glu Gly Phe Ser Pro Leu Leu Pro Val Gln Thr Ile Lys Glu Glu
485 490 495 500
gaa atc cag cct ggg gag gaa atg cca cac tta gcg aga ccc atc aaa 1831
Glu Ile Gln Pro Gly Glu Glu Met Pro His Leu Ala Arg Pro Ile Lys
505 510 515
gtg gag agc cct ccc ttg gaa gag tgg ccc tcc ccg gcc cca tct ttc 1879
Val Glu Ser Pro Pro Leu Glu Glu Trp Pro Ser Pro Ala Pro Ser Phe
520 525 530
aaa gag gaa tca tct cac tcc tgg gag gat tcg tcc caa tct ccc acc 1927
Lys Glu Glu Ser Ser His Ser Trp Glu Asp Ser Ser Gln Ser Pro Thr
535 540 545
cca aga ccc aag aag tcc tac agt ggg ctt agg tcc cca acc cgg tgt 1975
Pro Arg Pro Lys Lys Ser Tyr Ser Gly Leu Arg Ser Pro Thr Arg Cys
550 555 560
gtc tcg gaa atg ctt gtg att caa cac agg gag agg agg gag agg agc 2023
Val Ser Glu Met Leu Val Ile Gln His Arg Glu Arg Arg Glu Arg Ser
565 570 575 580
cgg tct cgg agg aaa cag cat cta ctg cct ccc tgt gtg gat gag ccg 2071
Arg Ser Arg Arg Lys Gln His Leu Leu Pro Pro Cys Val Asp Glu Pro
585 590 595
gag ctg ctc ttc tca gag ggg ccc agt act tcc cgc tgg gcc gca gag 2119
Glu Leu Leu Phe Ser Glu Gly Pro Ser Thr Ser Arg Trp Ala Ala Glu
600 605 610
ctc ccg ttc cca gca gac tcc tct gac cct gcc tcc cag ctc agc tac 2167
Leu Pro Phe Pro Ala Asp Ser Ser Asp Pro Ala Ser Gln Leu Ser Tyr
615 620 625
tcc cag gaa gtg gga gga cct ttt aag aca ccc att aag gaa acg ctg 2215
Ser Gln Glu Val Gly Gly Pro Phe Lys Thr Pro Ile Lys Glu Thr Leu
630 635 640
ccc atc tcc tcc acc ccg agc aaa tct gtc ctc ccc aga acc cct gaa 2263
Pro Ile Ser Ser Thr Pro Ser Lys Ser Val Leu Pro Arg Thr Pro Glu
645 650 655 660
tcc tgg agg ctc acg ccc cca gcc aaa gta ggg gga ctg gat ttc agc 2311
Ser Trp Arg Leu Thr Pro Pro Ala Lys Val Gly Gly Leu Asp Phe Ser
665 670 675
cca gta caa acc tcc cag ggt gcc tct gac ccc ttg cct gac ccc ctg 2359
Pro Val Gln Thr Ser Gln Gly Ala Ser Asp Pro Leu Pro Asp Pro Leu
680 685 690
ggg ctg atg gat ctc agc acc act ccc ttg caa agt gct ccc ccc ctt 2407
Gly Leu Met Asp Leu Ser Thr Thr Pro Leu Gln Ser Ala Pro Pro Leu
695 700 705
gaa tca ccg caa agg ctc ctc agt tca gaa ccc tta gac ctc atc tcc 2455
Glu Ser Pro Gln Arg Leu Leu Ser Ser Glu Pro Leu Asp Leu Ile Ser
710 715 720
gtc ccc ttt ggc aac tct tct ccc tca gat ata gac gtc ccc aag cca 2503
Val Pro Phe Gly Asn Ser Ser Pro Ser Asp Ile Asp Val Pro Lys Pro
725 730 735 740
ggc tcc ccg gag cca cag gtt tct ggc ctt gca gcc aat cgt tct ctg 2551
Gly Ser Pro Glu Pro Gln Val Ser Gly Leu Ala Ala Asn Arg Ser Leu
745 750 755
aca gaa ggc ctg gtc ctg gac aca atg aat gac agc ctc agc aag atc 2599
Thr Glu Gly Leu Val Leu Asp Thr Met Asn Asp Ser Leu Ser Lys Ile
760 765 770
ctg ctg gac atc agc ttt cct ggc ctg gac gag gac cca ctg ggc cct 2647
Leu Leu Asp Ile Ser Phe Pro Gly Leu Asp Glu Asp Pro Leu Gly Pro
775 780 785
gac aac atc aac tgg tcc cag ttt att cct gag cta cag tag 2689
Asp Asn Ile Asn Trp Ser Gln Phe Ile Pro Glu Leu Gln
790 795 800
agccctgccc ttgcccctgt gctcaagctg tccaccatcc cgggcactcc aaggctcagt 2749
gcaccccaag cctctgagtg aggacagcag gcagggactg ttctgctcct catagctccc 2809
tgctgcctga ttatgcaaaa gtagcagtca caccctagcc actgctggga ccttgtgttc 2869
cccaagagta tctgattcct ctgctgtccc tgccaggagc tgaagggtgg gaacaacaaa 2929
ggcaatggtg aaaagagatt aggaaccccc cagcctgttt ccattctctg cccagcagtc 2989
tcttaccttc cctgatcttt gcagggtggt ccgtgtaaat agtataaatt ctccaaatta 3049
tcctctaatt ataaatgtaa gcttatttcc ttagatcatt atccagagac tgccagaagg 3109
tgggtaggat gacctggggt ttcaattgac ttctgttcct tgcttttagt tttgatagaa 3169
gggaagacct gcagtgcacg gtttcttcca ggctgaggta cctggatctt gggttcttca 3229
ctgcagggac ccagacaagt ggatctgctt gccagagtcc tttttgcccc tccctgccac 3289
ctccccgtgt ttccaagtca gctttcctgc aagaagaaat cctggttaaa aaagtctttt 3349
gtattgggtc aggagttgaa tttggggtgg gaggatggat gcaactgaag cagagtgtgg 3409
gtgcccagat gtgcgctatt agatgtttct ctgataatgt ccccaatcat accagggaga 3469
ctggcattga cgagaactca ggtggaggct tgagaaggcc gaaagggccc ctgacctgcc 3529
tggcttcctt agcttgcccc tcagctttgc aaagagccac cctaggcccc agctgaccgc 3589
atgggtgtga gccagcttga gaacactaac tactcaataa aagcgaaggt ggacatgaaa 3649
aaaaaaaaaa aaaaaa 3665
<210> 75
<211> 801
<212> PRT
<213> Chile person
<400> 75
Met Lys Thr Ser Pro Arg Arg Pro Leu Ile Leu Lys Arg Arg Arg Leu
1 5 10 15
Pro Leu Pro Val Gln Asn Ala Pro Ser Glu Thr Ser Glu Glu Glu Pro
20 25 30
Lys Arg Ser Pro Ala Gln Gln Glu Ser Asn Gln Ala Glu Ala Ser Lys
35 40 45
Glu Val Ala Glu Ser Asn Ser Cys Lys Phe Pro Ala Gly Ile Lys Ile
50 55 60
Ile Asn His Pro Thr Met Pro Asn Thr Gln Val Val Ala Ile Pro Asn
65 70 75 80
Asn Ala Asn Ile His Ser Ile Ile Thr Ala Leu Thr Ala Lys Gly Lys
85 90 95
Glu Ser Gly Ser Ser Gly Pro Asn Lys Phe Ile Leu Ile Ser Cys Gly
100 105 110
Gly Ala Pro Thr Gln Pro Pro Gly Leu Arg Pro Gln Thr Gln Thr Ser
115 120 125
Tyr Asp Ala Lys Arg Thr Glu Val Thr Leu Glu Thr Leu Gly Pro Lys
130 135 140
Pro Ala Ala Arg Asp Val Asn Leu Pro Arg Pro Pro Gly Ala Leu Cys
145 150 155 160
Glu Gln Lys Arg Glu Thr Cys Ala Asp Gly Glu Ala Ala Gly Cys Thr
165 170 175
Ile Asn Asn Ser Leu Ser Asn Ile Gln Trp Leu Arg Lys Met Ser Ser
180 185 190
Asp Gly Leu Gly Ser Arg Ser Ile Lys Gln Glu Met Glu Glu Lys Glu
195 200 205
Asn Cys His Leu Glu Gln Arg Gln Val Lys Val Glu Glu Pro Ser Arg
210 215 220
Pro Ser Ala Ser Trp Gln Asn Ser Val Ser Glu Arg Pro Pro Tyr Ser
225 230 235 240
Tyr Met Ala Met Ile Gln Phe Ala Ile Asn Ser Thr Glu Arg Lys Arg
245 250 255
Met Thr Leu Lys Asp Ile Tyr Thr Trp Ile Glu Asp His Phe Pro Tyr
260 265 270
Phe Lys His Ile Ala Lys Pro Gly Trp Lys Asn Ser Ile Arg His Asn
275 280 285
Leu Ser Leu His Asp Met Phe Val Arg Glu Thr Ser Ala Asn Gly Lys
290 295 300
Val Ser Phe Trp Thr Ile His Pro Ser Ala Asn Arg Tyr Leu Thr Leu
305 310 315 320
Asp Gln Val Phe Lys Pro Leu Asp Pro Gly Ser Pro Gln Leu Pro Glu
325 330 335
His Leu Glu Ser Gln Gln Lys Arg Pro Asn Pro Glu Leu Arg Arg Asn
340 345 350
Met Thr Ile Lys Thr Glu Leu Pro Leu Gly Ala Arg Arg Lys Met Lys
355 360 365
Pro Leu Leu Pro Arg Val Ser Ser Tyr Leu Val Pro Ile Gln Phe Pro
370 375 380
Val Asn Gln Ser Leu Val Leu Gln Pro Ser Val Lys Val Pro Leu Pro
385 390 395 400
Leu Ala Ala Ser Leu Met Ser Ser Glu Leu Ala Arg His Ser Lys Arg
405 410 415
Val Arg Ile Ala Pro Lys Val Phe Gly Glu Gln Val Val Phe Gly Tyr
420 425 430
Met Ser Lys Phe Phe Ser Gly Asp Leu Arg Asp Phe Gly Thr Pro Ile
435 440 445
Thr Ser Leu Phe Asn Phe Ile Phe Leu Cys Leu Ser Val Leu Leu Ala
450 455 460
Glu Glu Gly Ile Ala Pro Leu Ser Ser Ala Gly Pro Gly Lys Glu Glu
465 470 475 480
Lys Leu Leu Phe Gly Glu Gly Phe Ser Pro Leu Leu Pro Val Gln Thr
485 490 495
Ile Lys Glu Glu Glu Ile Gln Pro Gly Glu Glu Met Pro His Leu Ala
500 505 510
Arg Pro Ile Lys Val Glu Ser Pro Pro Leu Glu Glu Trp Pro Ser Pro
515 520 525
Ala Pro Ser Phe Lys Glu Glu Ser Ser His Ser Trp Glu Asp Ser Ser
530 535 540
Gln Ser Pro Thr Pro Arg Pro Lys Lys Ser Tyr Ser Gly Leu Arg Ser
545 550 555 560
Pro Thr Arg Cys Val Ser Glu Met Leu Val Ile Gln His Arg Glu Arg
565 570 575
Arg Glu Arg Ser Arg Ser Arg Arg Lys Gln His Leu Leu Pro Pro Cys
580 585 590
Val Asp Glu Pro Glu Leu Leu Phe Ser Glu Gly Pro Ser Thr Ser Arg
595 600 605
Trp Ala Ala Glu Leu Pro Phe Pro Ala Asp Ser Ser Asp Pro Ala Ser
610 615 620
Gln Leu Ser Tyr Ser Gln Glu Val Gly Gly Pro Phe Lys Thr Pro Ile
625 630 635 640
Lys Glu Thr Leu Pro Ile Ser Ser Thr Pro Ser Lys Ser Val Leu Pro
645 650 655
Arg Thr Pro Glu Ser Trp Arg Leu Thr Pro Pro Ala Lys Val Gly Gly
660 665 670
Leu Asp Phe Ser Pro Val Gln Thr Ser Gln Gly Ala Ser Asp Pro Leu
675 680 685
Pro Asp Pro Leu Gly Leu Met Asp Leu Ser Thr Thr Pro Leu Gln Ser
690 695 700
Ala Pro Pro Leu Glu Ser Pro Gln Arg Leu Leu Ser Ser Glu Pro Leu
705 710 715 720
Asp Leu Ile Ser Val Pro Phe Gly Asn Ser Ser Pro Ser Asp Ile Asp
725 730 735
Val Pro Lys Pro Gly Ser Pro Glu Pro Gln Val Ser Gly Leu Ala Ala
740 745 750
Asn Arg Ser Leu Thr Glu Gly Leu Val Leu Asp Thr Met Asn Asp Ser
755 760 765
Leu Ser Lys Ile Leu Leu Asp Ile Ser Phe Pro Gly Leu Asp Glu Asp
770 775 780
Pro Leu Gly Pro Asp Asn Ile Asn Trp Ser Gln Phe Ile Pro Glu Leu
785 790 795 800
Gln
<210> 76
<211> 4
<212> PRT
<213> Artificial sequence
<220>
<223> Linker peptide
<400> 76
Asn Lys Arg Lys
1

Claims (19)

1. A peptide consisting of the amino acid sequence of SEQ ID NO. 56.
2. A polynucleotide encoding the peptide of claim 1.
3. A composition comprising a pharmaceutically acceptable carrier and at least one ingredient selected from the following groups (a) to (e):
(a) The peptide of claim 1;
(b) A polynucleotide encoding in expressible form the peptide of claim 1;
(c) An Antigen Presenting Cell (APC) that presents on its cell surface a complex of the peptide of claim 1 and HLA-A 01;
(d) Exosomes presenting on their cell surfaces the complex of the peptide of claim 1 with HLA-A01, and
(E) A CTL, which targets the peptide of claim 1.
4. A composition according to claim 3, which is a composition for inducing CTLs, wherein the ingredient is at least one ingredient selected from the following groups (a) to (d):
(a) The peptide of claim 1;
(b) A polynucleotide encoding in expressible form the peptide of claim 1;
(c) An Antigen Presenting Cell (APC) which presents on its cell surface a complex of the peptide of claim 1 and HLA-A01, and
(D) Exosomes presenting on their cell surfaces the complex of the peptide of claim 1 with HLA-A 01.
5. The composition of claim 3 which is a pharmaceutical composition.
6. The composition of claim 5 for use in one or more selected from the group consisting of (i) cancer treatment, (ii) cancer prevention (prophltaxis) and (iii) prevention (prevention) of postoperative cancer recurrence.
7. The composition of claim 5 for inducing an immune response against cancer.
8. The composition of claim 6, wherein the cancer is selected from the group consisting of Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myelogenous Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor, and testicular tumor.
9. The composition of any one of claims 3 to 8, formulated for administration to a subject that is positive for at least HLA-A 01.
10. A method of inducing APC having CTL inducibility comprising the step of:
(a) Contacting an APC in vitro or ex vivo with a peptide according to claim 1, and
(B) Introducing a polynucleotide encoding the peptide of claim 1 into an APC in vitro or ex vivo.
11. An in vitro method of inducing CTLs comprising the steps selected from the group consisting of:
(a) Co-culturing CD8 positive T cells with APCs that present on their surface a complex of HLA antigens and the peptide of claim 1;
(b) Co-culturing CD8 positive T cells with exosomes presenting complexes of HLA antigens with the peptide of claim 1 on their surface, and
(C) Introducing a polynucleotide encoding each subunit of a T Cell Receptor (TCR) capable of binding to the peptide of claim 1 presented on the cell surface by HLA antigens into CD8 positive T cells.
An apc which presents on its surface a complex of an HLA antigen and the peptide of claim 1.
13. The APC of claim 12, induced by the method of claim 10.
Ctl, which targets the peptide of claim 1.
15. The CTL of claim 14, which is induced by an in vitro method comprising a step selected from the group consisting of:
(a) Co-culturing CD8 positive T cells with APCs that present on their surface a complex of HLA antigens and the peptide of claim 1;
(b) Co-culturing CD8 positive T cells with exosomes presenting complexes of HLA antigens with the peptide of claim 1 on their surface, and
(C) Introducing a polynucleotide encoding each subunit of a T Cell Receptor (TCR) capable of binding to the peptide of claim 1 presented on the cell surface by HLA antigens into CD8 positive T cells.
16. An emulsion comprising the peptide of claim 1, a water-soluble carrier and an oil adjuvant.
17. A kit comprising a container containing the composition of any one of claims 3 to 9 and a container containing an adjuvant.
18. Use of an ingredient for the preparation of a pharmaceutical composition for inducing an immune response against FOXM 1-expressing cancer, wherein the ingredient is selected from the group consisting of (a) - (e) below:
(a) The peptide of claim 1;
(b) A polynucleotide encoding in expressible form the peptide of claim 1;
(c) APC, which presents on its cell surface a complex of the peptide of claim 1 with HLA-A 01;
(d) Exosomes presenting on their cell surfaces the complex of the peptide of claim 1 with HLA-A01, and
(E) A CTL targeting the peptide of claim 1, wherein said cancer is selected from the group consisting of Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myeloid Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor and testicular tumor.
19. Use of an ingredient for the preparation of a pharmaceutical composition for the treatment and/or prevention of FOXM1 expressing cancers, and/or for the prevention of postoperative recurrence thereof, wherein the ingredient is selected from the group consisting of (a) - (e) below:
(a) The peptide of claim 1;
(b) A polynucleotide encoding in expressible form the peptide of claim 1;
(c) APC, which presents on its cell surface a complex of the peptide of claim 1 with HLA-A 01;
(d) Exosomes presenting on their cell surfaces the complex of the peptide of claim 1 with HLA-A01, and
(E) A CTL targeting the peptide of claim 1, wherein said cancer is selected from the group consisting of Acute Myelogenous Leukemia (AML), bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic Myeloid Leukemia (CML), colon cancer, esophageal cancer, gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, small Cell Lung Cancer (SCLC), soft tissue tumor and testicular tumor.
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